The Anionic Phospholipids in the Plasma Membrane Play an

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The anionic phospholipids in the plasma membrane play an important role in regulating the biochemical properties and biological functions of RecA proteins Deepika Prasad, and Kalappa Muniyappa Biochemistry, Just Accepted Manuscript • DOI: 10.1021/acs.biochem.8b01147 • Publication Date (Web): 06 Feb 2019 Downloaded from http://pubs.acs.org on February 7, 2019

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Biochemistry P a g e |1

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The anionic phospholipids in the plasma membrane play an important role in

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regulating the biochemical properties and biological functions of RecA proteins

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Deepika Prasad and Kalappa Muniyappa1

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Department of Biochemistry, Indian Institute of Science, Bengaluru 560012, India

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1To

whom correspondence should be addressed: e-mail: [email protected] Tel: (91-80) 2293 2235/2360 0278; Fax: (91-80) 2360 0814/0683

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Running title: Anionic phospholipids regulate RecA functions

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Key words: Mycobacteria, Anionic phospholipids, plasma membrane, RecA nucleoprotein

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filaments, DNA strand exchange, SOS response

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ABSTRACT

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Escherichia coli RecA (EcRecA) forms discrete foci that cluster at cell poles during

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normal growth, which are redistributed along the filamented cell axis upon induction of the

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SOS response. The plasma membrane is thought to act as a scaffold for EcRecA foci, thereby

15

playing an important role in RecA-dependent homologous recombination. Further, in vivo

16

and in vitro studies demonstrate that EcRecA binds strongly to the anionic phospholipids.

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However, there have been almost no data on the association of mycobacterial RecA proteins

18

with the plasma membrane and the effects of membrane components on their function. Here,

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we show that mycobacterial RecA proteins specifically interact with phosphatidylinositol and

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cardiolipin among other anionic phospholipids; however, they had no effect on the ability of

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RecA proteins to bind single-stranded DNA. Interestingly, phosphatidylinositol and

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cardiolipin impede the DNA-dependent ATPase activity of RecA proteins, although ATP-

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binding is not affected. Furthermore, the ability of RecA proteins to promote DNA strand

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exchange is not affected by anionic phospholipids. Strikingly, anionic phospholipids suppress

25

the RecA-stimulated autocatalytic cleavage of the LexA repressor. The Mycobacterium

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smegmatis RecA foci localize to the cell poles during normal growh, and these structures

27

disassemble and reassemble into several foci along the cell after DNA damage induction.

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Taken together, these data support the notion that the interaction of RecA with cardiolipin

29

and phosphatidylinositol, the major anionic phospholipids of the mycobacterial plasma

30

membrane, may be physiologically relevant, as they provide a scaffold for RecA storage and

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may regulate recombinational DNA repair and the SOS response.

32 33

INTRODUCTION

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RecA, a protein having multifaceted biological functions, is among the most conserved

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proteins across bacterial organisms.1, 2 It plays a pivotal role in the repair of stalled replication

36

forks, double-strand break (DSB) repair, homologous recombination, and the SOS response.3-6

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Along with several other accessory proteins, RecA protein promotes the pairing of homologous

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DNA molecules and DNA strand exchange, coupling ATP hydrolysis to the branch movement in

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the extension phase, and the autocatalytic cleavage of LexA, UmuD and related phage

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repressors.1,7 A critical step in these processes is the cooperative binding of RecA to ssDNA

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resulting in the formation of a right-handed RecA/ssDNA nucleoprotein filament.8-10

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Accumulating evidence suggests that in Escherichia coli and Bacillus subtilis, RecA forms an

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array of long parallel bundle-like structures that are located underneath the inner membrane in

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living cells before and after DNA damage induction.11-16 Furthermore, these bundles are

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proposed to mediate a search for DNA sequence homology and homologous pairing between

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distant sisters during the repair of double-strand breaks.11,17 By contrast, a recent study shows


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that repair of a replication-dependent DSB involves a transient RecA focus, but not the formation

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of RecA bundles.18

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The pioneering studies of E. Witkin and A Pardee led to the discovery that increased

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amounts of RecA becomes associated with the membrane fractions of SOS induced Escherichia

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coli cells.19-22 More broadly, these findings indicate that the association of RecA with the cell

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membrane may regulate RecA-dependent SOS induction, SOS mutagenesis, DNA replication

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and homologous recombination.1,7 While the association of E. coli RecA with the membrane

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fraction has long been recognized, the identity of the components and their significance remains

55

unclear. Why might RecA interact with the cell membrane, and what is its physiological

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significance, especially with regard to recombinational DNA repair? An early study showed that

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E. coli RecA binds specifically to anionic phospholipids such as cardiolipin (CL) and

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phosphatidylglycerol (PG), and that the interaction leads to the inhibition of RecA binding to

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single-stranded DNA.23 In recent studies, the N-terminal helix and L2 loop of RecA have been

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shown to interact tightly with PG and CL.12, 24

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The unique cell envelopes of mycobacteria are critical for their physiology and host-

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pathogen interface because they provides a formidable barrier against antibiotics and also serve

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as a major virulence determinants.25-27 The cell walls of mycobacteria are rigid and very complex

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structures that are extremely rich in glycolipids, including mycolic acids, phosphatidyl inositol

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mannosides, phthiocerol dimycocerosates and lipoglycans,25,27-31 while, PI, CL and

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phosphatidylethanolamine form non-bilayer structures.25,28-31 One of the physiological hallmarks

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of M. tuberculosis-infected macrophages is the generation of reactive oxygen species and nitric

68

oxide, which are likely to induce DNA damage, thereby severely compromising their

69

intracellular

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recombination/repair machinery for the maintenance and repair of its genome. It is also

survival.

Therefore,

the

pathogen

must


possess

more

robust

DNA

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conceivable that biological processes involving cellular membrane components play crucial

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regulatory roles in the function of mycobacterial RecA proteins.

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The vital question that is addressed in this study is how the interaction with anionic

74

phospholipids affects the function of mycobacterial RecA proteins. We found that anionic

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phospholipid components that are found in the plasma membrane of mycobacteria play an

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important role in regulating the function of M. smegmatis RecA and M. tuberculosis RecA

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proteins (henceforth referred to as MsRecA and MtRecA). These results are consistent with a

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model wherein mycobacteria employ lipid-based mechanisms to regulate the levels of free RecA

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in the living cell as well as impede RecA catalysed ATP hydrolysis and autocatalytic cleavage of

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LexA, thereby protecting the genome against the fortuitous activation of SOS-induced

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mutagenesis. In contrast to the scenario in E. coli and B. subtilis,11,15 Mycobacterium smegmatis

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RecA foci localize to the cell poles during normal metabolism, and these structures disassemble

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and reassemble into several foci along the cell after DNA damage induction. In addition, the role

84

of anionic phospholipids on E. coli RecA (EcRecA) and mycobacterial RecAs are compared to

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comprehensively investigate the effects on RecA function.

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EXPERIMENTAL PROCEDURES

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Biochemicals, proteins and DNA

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All the chemicals were of analytical grade. These were obtained from GE Healthcare

90

Life Sciences (Piscataway, NJ) or Sigma-Aldrich (St. Louis, MO). T4 polynucleotide kinase

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was purchased from Thermo Scientific (Waltham, MA). The oligonucleotides (Table 1) were

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procured from Sigma-Genosys (Singapore). FAM-labelled oligonucleotide (ODN3) was

93

obtained from Integrated DNA Technologies (Skokie, Illinois). [γ-32P]ATP was purchased

94

from the Board of Radiation and Isotope Technology (Hyderabad, India) and dATP from GE


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Healthcare Life Sciences (Piscataway, NJ). The polyethylenimine cellulose TLC plates,

96

ATPγS, phosphocreatine, creatine phosphokinase, 1,4-diazabicyclo[2.2.2]octane (DABCO),

97

4',6-diamidino-2-phenylindole (DAPI), phospholipids and polyclonal antibodies against

98

GroEL and YME1L1 were obtained from Sigma-Aldrich (St. Louis, MO). The full-length M.

99

tuberculosis RecA (MtRecA), M. smegmatis RecA (MsRecA) and E. coli RecA (EcRecA)

100

proteins, devoid of any affinity tags, were purified as described.32, 33

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Bacterial strains, plasmids and growth conditions

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A single colony of M. smegmatis mc2155 from a Middlebrook 7H10 agar plate

103

containing 10% ADC (albumin-dextrose-catalase) was inoculated into a Middlebrook 7H9

104

liquid medium supplemented with 0.05% Tween 80 and 10% ADC. The cells were grown at

105

37 °C for 2 days under 120 rpm agitation. M. smegmatis mc2155 strain was used for RecA-

106

GFP fluorescence assays.34 The recA gene was cloned in a pJB(-) vector (a gift from Mary

107

Jackson, Colorado State University, USA) which harbours a GFP tag at the C-terminus. The

108

electrocompetent cells were prepared from cultures grown in a Middlebrook 7H9 liquid

109

medium supplemented with Tween 80 and 10% ADC to A600 of 0.8. The cells were washed

110

and resuspended in 10% chilled glycerol prior to use. The RecA-GFP construct and the

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empty vector (pJB (-)) were transformed by electroporation into M. smegmatis mc2155

112

competent cells and were selected on Middlebrook 7H10 agar containing kanamycin (50

113

µg/ml).35 The visualization of RecA-GFP was carried out using fluorescence microscopy.

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The cells equivalent to 0.6 optical density at A600 were exposed to UV irradiation (254 nm) at

115

a dose of 25 J/m2 to induce DNA damage.

116

Preparation of radiolabeled DNA substrates

117

The oligonucleotide, ODN2, was radiolabeled at the 5′ end by using [γ-32P]ATP and T4

118

polynucleotide kinase.36 The double-stranded DNA (83 bp) was prepared by annealing

119

labeled ODN2 with unlabeled ODN1 at a ratio of 1:1 in a buffer (120 μl) containing 10 mM


32P-

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Tris-HCl (pH 7.5), 1 mM EDTA, and a 1X SSC solution (150 mM NaCl and 15 mM sodium

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citrate). The hybridization mixture was incubated at 95 °C for 5 min and was followed by

122

gradual cooling to 4 °C. The annealed

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separated by electrophoresis on a 10% polyacrylamide gel in a 44.5 mM Tris-borate buffer

124

(pH 8.3) containing 1 mM EDTA at 150 V for 7 h. The bands corresponding to ssDNA and

125

dsDNA were excised from the gel, and eluted into a TE buffer (10 mM Tris–HCl, pH 7.5, 1

126

mM EDTA). The concentrations of unlabeled and radiolabeled DNA substrates were

127

expressed in moles of nucleotide residues/liter.

32P-labeled

dsDNA and

32P-labeled

ssDNA were

128 129

Table 1. Sequence of oligonucleotides used in this study.

130 Oligonucleotide ODN1

Sequence (5′→3′) GATCTGTACGGCTGGACAGTGTTGTGAGTGAG

Length 83

TTGAAGATGGGAGGTAGTGTGCTAGGTGGCTT AGGAGAGAGTCGTTAGTGT ODN2

ACACTAACGACTCTTCCTAAGCCACCTAGCAC

83

ACTACCTCCCATCTTCAACTCACTCACAACAC TGTCCAGCCGTACAGATC ODN3

GCAGATCTGGCCTGATTGCGGTAGA

50

GATGGAGCCGTAACAGTACGTAGTC-6-FAM 131 132

Preparation of liposomes

133

The liposomes were prepared using different phospholipids either alone or in

134

combination with cardiolipin as described.12 The indicated phospholipid (1 mg) was

135

dissolved in a 200 µl solvent (chloroform-methanol at a ratio of 1:10). The solvent was


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evaporated to form a film in a glass vial (5 ml) under a vacuum. The dry lipid material was

137

rehydrated with sterilized MilliQ water overnight at 4 °C. The suspension (5 mM) was first

138

incubated at 55 °C for 30 min, vortexed for 2 min, then incubated again for 2 min on ice. This

139

step was repeated 5 times. Finally, the suspension was subjected to sonication at 40 °C for 30

140

min. The liposomes having the following phospholipid composition were used in this study:

141

PI, PC, DOPE, PG (100%), PI:CL, DOPE:CL, PC:CL, PG:CL (70:30 %). Unless otherwise

142

mentioned, the ratios correspond to weight percentages.

143

Dynamic light scattering

144

Dynamic light scattering (DLS) measurements were performed using diluted samples

145

(0.01 mg/ml) to determine the distribution of the size of the liposome particles on a

146

SpectroSize 300 instrument (Molecular Dimension Ltd., Suffolk, UK) at 660 nm as

147

described.37 The scattered light was measured at an angle of 90°. Each sample was subjected

148

to 25 readings. The data were analyzed using the SpectroSize 300 software to obtain the

149

polydispersity profiles of the samples.

150

Co-flotation assay

151

The co-flotation assay was performed as described.12 The RecA from the indicated

152

source was incubated with different liposomes (PS, PC, PI, DOPE, PI:CL and DOPE:CL) at

153

37 °C for 5 min and then mixed with 80% of Accudenz (Accurate Chemical and Scientific

154

Corporation, Westbury, NY), prepared in 125 mM HEPES, 500 mM KCl and 50% glycerol.

155

The resulting mixture (40% Accudenz solution) was transferred to a polycarbonate

156

ultracentrifuge tube (Beckman Coulter, Bangalore). A gradient was formed by layering

157

successive decreasing densities of Accudenz solutions (35%, 30% and 0%). The gradients

158

were centrifuged at 281227 × g at 4 °C for 2 h in a Beckman Coulter Optima Max XP table-

159

top ultracentrifuge with a TLA 120.2 rotor. After centrifugation, aliquots (20 µl) from the top


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layer were analysed by 12.5% denaturing SDS-PAGE. The proteins were visualized by silver

161

staining.

162

Protein-lipid overlay assay

163

The assay was performed as described.38 The serial dilutions of liposomes were spotted

164

onto nitrocellulose membranes and blocked by incubation with 3% fatty acid free bovine

165

serum albumin (BSA) in a TBST buffer [50 mM Tris-HCl (pH 7.5), 150 mM NaCl and 0.1%

166

Tween 20 (w/v)] for 1 h. The membranes were treated with a blocking buffer containing 10

167

µg RecA/ml at 24 °C for 1 h. The membrane was washed extensively with TBST buffer over

168

a period of 45 min, followed by incubation with an anti-RecA antibody (1:15000 dilution) for

169

6 h at 4 °C with gentle shaking. After washing the membrane with the TBST buffer, three

170

times of 10 min each, a horseradish peroxidase-conjugated anti-rabbit secondary antibody

171

(Sigma-Aldrich, St. Louis, MO, USA) was added and incubation was continued for 4 h at 4

172

°C. The blots were incubated with an anti-horseradish peroxidase conjugated antibody and

173

the proteins were detected by enhanced chemiluminescence (Merck Millipore, Bangalore).

174

The fluorescent protein bands were imaged using a ChemiDoc ImageQuant LAS 4000 Mini

175

apparatus (GE Healthcare Life Sciences Imaging System, Bangalore).

176

Tryptophan fluorescence measurements

177

The assay was carried out as described.12 The reaction mixture (110 µl) containing an

178

assay buffer (20 mM Tris-acetate, pH 7.5, 20% glycerol, 0.1 mM EDTA, 1 mM DTT) and

179

0.5 µM RecA was incubated at 37 °C for 5 min. The tryptophan residues (Trp 290 and 308 in

180

E. coli RecA and Trp 290 in mycobacterial RecA proteins) were excited at 295 nm and the

181

emission spectra were recorded from 320 to 420 nm at a slit width of 5 nm. The assay was

182

carried out as a function of increasing concentrations of liposomes containing 100% PG, PI,

183

PE, PC; 70%:30% PI:CL or 70%:30% PE:CL. To obtain information about the affinity (Kd


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values) of RecA for different liposomes, a fixed concentration of the indicated protein was

185

titrated against increasing concentrations of liposomes.

186

Fluorescence anisotropy measurements

187

The assay was performed as described.12,39 Ten nM fluorescein (FAM)-labeled 50-mer

188

ssDNA (ODN3) was incubated in a buffer (500 µl) containing 25 mM Tris-HCl (pH 7.5), 10

189

mM MgCl2, 5% glycerol, 1 mM DTT, 1 mM ATPγS and the indicated concentrations (30, 60,

190

100, 160, 200, 300, 500 and 1000 nM) of RecA at 37 °C for 20 min in the dark. The

191

anisotropy data were collected using an excitation wavelength of 490 nm and emission at 520

192

nm at 25 °C on a FluoroLog-3 spectrofluorometer (Horiba Scientific, Edison, NJ). The data

193

were analyzed by plotting anisotropy levels against increasing concentrations of RecA. The

194

data were fitted into one site-specific binding equation Y = Bmax*X/(Kd + X) to determine the

195

Kd values, where Bmax is the maximum specific binding and Kd is the equilibrium binding

196

constant. In a separate experiment, the anisotropy levels were recorded by challenging the

197

preformed RecA nucleoprotein filaments (under the conditions as described above) with the

198

addition of increasing concentrations of PI (10, 50, 100, 200, 500 and 1000 µM). The

199

changes in the levels of anisotropy were plotted as a function of increasing concentrations of

200

phosphatidylinositol using GraphPad Prism 5 software.

201

Electrophoretic mobility shift assay

202

The assay was performed in a reaction mixture (20 μl) containing 20 mM Tris-HCl (pH32P-labeled

203

7.5), 8 mM MgCl2, 1 mM dATP, 1 mM DTT, 1 μM

ssDNA and 1 µM RecA.

204

Increasing concentrations of liposomes (25, 50, 75, 100, 200, 400, 800 and 1000 µM) and

205

32P-labelled

206

indicated RecA protein. After incubation at 37 °C for 20 min, the reaction was terminated by

207

the addition of a 2 μl gel-loading dye. The samples were separated on a 8% native

ssDNA (83 mer) were incubated at 37 °C for 5 min prior to the addition of the


Biochemistry 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

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208

polyacrylamide gel in a TBE buffer [44.5 mM Tris-borate buffer (pH 8.3) containing 1 mM

209

EDTA] at 10 V/cm at 4 °C for 4 h. The dried gels were exposed to the phosphorimaging

210

screen and the images were acquired using a Fuji FLA-9000 phosphorImager.

211

ATPase assay

212

The ATPase assay was performed as described.33,40 The reaction mixture (20 µl)

213

containing 20 mM Tris-HCl (pH 7.5), 1 mM DTT, 8 mM MgCl2, 10 μM M13 ssDNA, and 1

214

μM RecA was incubated with increasing concentrations of liposomes (10, 20, 30, 40, 50, 60,

215

80 and 100 µM) for 10 min at 37 °C. The reaction was initiated by the addition of 10 µM [γ-

216

32P]ATP

217

addition of 25 mM EDTA. Two l aliquots were spotted on a PEI-cellulose TLC plate. After

218

drying, the chromatogram was placed in a glass solvent tank containing 0.5 M LiCl and 1 M

219

formic acid. The solvent front was allowed to migrate to a position near the top of the TLC

220

plate. The TLC plate was air-dried after its removal from the tank and the bands were

221

visualized using a Fuji FLA-9000 phosphorimager.

222

ATP binding assay

followed by incubation for 30 min at 37 °C. The reaction was stopped by the

223

The assay was performed as described.33 The indicated RecA (2.5 µM) was preincubated

224

with liposomes of different anionic phospholipids (10, 25, 50, 75 or 100 µM) at 37 °C for 10

225

min. The binding reaction was initiated by the addition of 10 µM [γ-32P]ATP and incubation

226

was continued at 37 °C for 30 min. The reaction mixtures were irradiated on ice with UV

227

light (254 nm) in a Stratalinker (Stratagene California, San Diego, CA) for 5 min. After the

228

addition of a Laemmli buffer (5 µl) followed by incubation at 95 °C for 5 min, the samples

229

were separated on a 12.5% SDS-polyacrylamide gel. The dried gel was exposed to the

230

phosphorimaging

231

phosphorImager.

screen

and

the

images

were

acquired


using

Fuji

FLA-9000

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Biochemistry P a g e | 11

232

DNA strand exchange assay

233

The assay was performed as described.32 The reaction mixtures (10 μl) containing 20

234

mM Tris–HCl pH 7.5, 3 mM dATP, 8 mM MgCl2, 5 μM unlabeled ssDNA (83 mer), 2.5 μM

235

MsRecA were incubated in the presence of an ATP regeneration system (5 mM

236

phosphocreatine and 10 U/ml creatine phosphokinase) at 37 °C for 5 min. Subsequently,

237

increasing concentrations of liposomes were added (25, 50, 75, 100, 125, 150, 175, 200 and

238

250 µM) and incubation was extended for 5 min. The reaction was initiated by the addition of

239

1 μM 32P-labeled dsDNA (83 bp) and incubation was continued at 37 °C for an additional 20

240

min. The reaction was terminated by the addition of a 2.5 μl 5X stop solution (5% SDS and

241

100 mM EDTA) and 1 μl of proteinase K (10 mg/ml), followed by incubation at 37 °C for 10

242

min. After the addition of a 1.4 μl 10X gel loading solution [50% glycerol, 0.42% (w/v)

243

bromophenol blue, 0.42% (w/v) xylene cyanol], the samples were loaded onto a 8%

244

polyacrylamide gel and electrophoresed in a 44.5 mM Tris-borate buffer (pH 8.3) containing

245

1 mM EDTA at 150 V for 6 h. The dried gels were exposed to the phosphorimaging screen

246

and the images were acquired using a Fuji FLA-9000 phosphorImager.

247

Coprotease assay

248

The assay was performed as described.41 The reaction mixtures contained 20 mM Tris-

249

HCl (pH 7), 8 mM MgCl2, 1 mM DTT, 3 mM dATP, 5 µM ssDNA and 2.5 µM MsRecA,

250

MtRecA or EcRecA. After preincubation for 5 min at 30 °C, the reaction was initiated by the

251

addition of 5 µM LexA and incubation was extended at 30 °C for 30 min. The reaction was

252

terminated by the addition of Laemmli's sample buffer.42 The samples were incubated at 95

253

°C for 5 min and then separated on a 12.5% SDS-polyacrylamide gel. The bands were

254

visualized using silver staining.

255

Genomic DNA isolation and gene amplification


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256

M. smegmatis mc2155 genomic DNA was isolated as described.43 DNA manipulations

257

were conducted according to standard procedures.36 Plasmid DNA and the desired DNA

258

fragments were isolated from the gels using a Qiagen Miniprep kit according to

259

manufacturer’s instructions. M. smegmatis genomic DNA was PCR amplified as described.44

260 261

Construction of GFP-tagged RecA

262

The M. smegmatis recA gene (MSMEG_2723) sequence was obtained from the

263

SmegmaList database (http://svitsrv8.epfl.ch/mycobrowser/smegmalist.html). The recA gene

264

was

265

AACGCATATGGCGCAGCAGGCCCCAGATC-3′)

266

TTACGAATTCGAAGTCAACCGGGGCCGGG-3′) primers harbouring NdeI and EcoRI

267

recognition sequences (underlined). The PCR product and the cloning vector were digested

268

with NdeI and EcoRI. The gel purified PCR product was ligated into a pJB(-) expression

269

vector at the NdeI and EcoRI site of the multiple cloning site. The resulting plasmid

270

contained a full-length M. smegmatis recA gene fused to a GFP coding sequence at the C-

271

terminus.

272

Microscopy and image analysis

PCR

amplified

using

specific

forward

(5′-

and

reverse

(5′

273

A single M. smegmatis mc2155 colony harbouring either empty vector pJB(-) or an

274

RecA-GFP construct was grown in a Middlebrook 7H9 liquid medium containing 50 µg/ml

275

kanamycin supplemented with Tween 80 and 10% ADC as a starter culture. The secondary

276

culture (10 ml) was grown until O.D.600 ~0.6 and then exposed to UV radiation (25 J/m2). The

277

untreated and UV-treated cells were further grown in the dark for 3 h at 37 °C. The cells

278

(from 1 ml culture) were collected by centrifugation at 6000 rpm for 10 min and washed with

279

phosphate buffered saline (PBS). The pellet was resuspended in 200 µl PBS and incubated


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280

with a 4',6'-diamidino-2-phenylindole (DAPI) solution (10 µg/ml) for 20 min at 24 °C. The

281

cells were washed twice and resuspended in PBS. An aliquot (5-10 µl) was spread evenly on

282

the glass slide. After air-drying, 5 µl of an antifade reagent (1% DABCO in 90% glycerol)

283

was placed over the sample, followed by a glass cover over the specimen. The sealed slides

284

were stored in the dark at 4 °C. The confocal images were acquired on a Zeiss confocal

285

microscope (Carl Zeiss, Germany) using a Plan-Apochromat 63X oil immersion objective.

286

Subcellular fractionation

287

The isolation of membrane and cytoplasmic fractions was carried out as described.45

288

M. smegmatis mc2155 was grown in 150 ml of a Middlebrook 7H9 broth containing 0.2%

289

glycerol and 0.05% Tween 80 at 37 °C, 120 rpm until O.D.600 ~ 0.8. Damage to genomic

290

DNA was induced by the addition of mitomycin C (200 ng/ml). The cells from untreated and

291

mitomycin C - treated cultures (20 ml) at different time points were harvested by

292

centrifugation at 12000×g for 10 min. The pellets were washed twice with buffer A (50 mM

293

Tris-HCl pH 7.4, 50 mM KCl, 10% glycerol, 0.5% NP-40 and 1 mM PMSF). The cells were

294

resuspended in 1 ml of buffer A and lysed by sonication at an amplitude of 21%. Each pulse

295

consisted of 1 sec and 30 sec, which was repeated 6 times. The cell debris was removed by

296

centrifugation at 17000×g for 10 min at 4 °C. The supernatant was centrifuged at 100000×g

297

for 2.5 h at 4 °C. The pellet was solubilized with 0.5 ml of buffer B (1 M NaCl, 10%

298

glycerol, 0.5% NP-40 and 1 mM PMSF) for 6 h at 4 °C. The suspension was subjected to

299

centrifugation at 100000×g at 4 °C for 2.5 h. The pellet was solubilized in 150 µl of buffer A

300

for 6 h at 4 °C. The insoluble material was removed by centrifugation at 12000×g for 10 min

301

at 4 °C. The supernatant was used as the source of a pure membrane fraction.

302

Western blotting


Biochemistry 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 14 of 51 P a g e | 14

303

The whole cell lysates from mitomycin C-treated and untreated cultures of M.

304

smegmatis mc2155 at different time points were fractionated into cytoplasmic and membrane

305

fractions as described above. The polyclonal antibodies against GroEL and YME1L1 (human

306

ortholog of FtsH which cross reacts with M. smegmatis FtsH) were purchased from Sigma-

307

Aldrich (St. Louis, MO, USA) and the polyclonal antibody against M. smegmatis RecA was

308

raised in rabbits and characterized as described.46 Equal amounts of the total protein from

309

cytosol and membrane fractions (30 and 15 µg respectively) were boiled in SDS-PAGE

310

buffer for 10 min and separated by 10% SDS-PAGE as described.42 After electrophoresis, the

311

proteins were transferred onto a PVDF membrane using a semi-dry transfer apparatus (GE

312

Healthcare Life Sciences, Piscataway, NJ). The membranes were blocked with 3% BSA in

313

PBS. The membranes were treated with polyclonal antibodies against RecA (1:15000),

314

GroEL (1:80000) and FtsH (1:1500). The membranes were washed with TBST (50 mM Tris-

315

HCl pH 8.0, 150 mM NaCl and 1% Tween 20) and then incubated with a peroxidase-

316

conjugated secondary anti-rabbit antibody. The blots were developed using luminol and

317

hydrogen peroxide. The fluorescent protein bands were imaged using a Quant LAS-4000

318

chemidocumentation system (GE Life Science, PA, USA), and quantified by using ImageJ

319

software.

320 321

RESULTS

322

Characterization of liposomes

323

In this study, liposomes consisting of phosphatidylinositol and cardiolipin (PI:CL

324

70:30, w/v) were prepared as model membranes to test their effect on RecA protein-

325

dependent functions. The liposomes were prepared by hydrating the dry lipid films followed

326

by direct sonication of anionic phospholipids in an aqueous medium. The particle size and


Page 15 of 51 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


327

polydispersity index were determined by DLS measurements (Figure 1A, 1D). The liposomes

328

of different phospholipid compositions were in the size range of 90-160 nm (Fig. 1E). The

329

polydispersity index of below 20% indicated that the phospholipid liposomes were

330

homogeneous. To carry out the experiments described below, we used these liposomes,

331

hereinafter referred to with their respective phospholipid constituents.

332 333

Figure 1. The particle size and polydispersity index of liposomes. The liposomes were

334

characterized for their size (radius in nm) and homogeneity in terms of their polydispersity

335

index. Panels A-D illustrate the data for one of the liposomes (PI:CL,70:30, w/v). (C) and

336

(D): The graphs show the size distribution of liposome in different forms. (E) Table shows

337

the size of the liposomes and the polydispersity index (%) of the liposomes used in this study.

338

The figure (A-D) are representative of three independent experiments.

339

RecA interacts with liposomes containing phospholipids

340

We first sought to determine the ability of MsRecA to interact with phospholipids. In

341

these experiments, nitrocellulose strip assay was used as an initial screen to investigate the

342

lipid binding properties of MsRecA. The assay was carried out by spotting increasing

343

concentrations of lipid vesicles of defined composition on nitrocellulose membranes. The


Biochemistry 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 16 of 51 P a g e | 16

344

membranes were blocked with a solution containing 3% fatty acid free BSA and then

345

incubated with MsRecA. The interaction between MsRecA and the phospholipid was

346

detected by Western blotting using MsRecA-specific antibodies. Figure 2 demonstrates that

347

MsRecA bound to multiple acidic phospholipids in a dose-dependent manner. Although

348

MsRecA bound with higher affinity to PI, PG and PE:CL, interactions with DOPE and a

349

mixture of PI:CL was also observed, thereby supporting the idea that MsRecA can bind to

350

both simple and complex phospholipids. However, MsRecA did not interact with every

351

phospholipid tested in the strip assay (e.g., PC or PS).

352 (A)

(C)

(B)

PI

1

1

2

3

2

4

3

5 6 PI:CL (70:30)

4

5

(E)

2

3

4

1 2

(D)

3

1

6

4

2

3

5

5 6 PE:CL (70:30)

4

(F)

PG

1

DOPE

5

6

PS

1

6

(G)

2

3

4

5

6

PC

1

2

3

4

5

6

353 354 355

Figure 2. The protein-lipid overlay assay shows that RecA binds to phospholipids. The assay

356

was performed using liposomes prepared from different phospholipids and MsRecA. Panels

357

A-G: spot 1, BSA; 2, M. tuberculosis RecX; 3-6, increasing concentrations of phospholipids

358

(0.5, 0.75, 1 and 1.5 µg) spotted on a nitrocellulose membrane (indicated by the filled

359

rectangle at the top of each panel). After blocking the membrane with non-fatty acid BSA,

360

MsRecA was incubated with the membrane and the interactions were probed using RecA-


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361

specific antibodies as described in Experimental Procedures. The figure shown is a

362

representative of three independent experiments.

363

Although the strip assay is a simple and most commonly used type of test to screen for

364

lipid-binding properties of proteins, it should be noted that it is prone to false positives or

365

false negatives, and thus may not accurately report on the specificity of MsRecA-lipid

366

interactions. Therefore, a more physiologically relevant liposome co-flotation binding assay

367

was performed to monitor the liposome binding activity of RecA proteins. The lipid vesicles

368

of defined composition were mixed with RecA from the specified bacterial species. An

369

aliquot of liposome-RecA suspension was placed under a discontinuous Accudenz gradient

370

[35%, 30% and 0% (v/v)]. After ultracentrifugation, the liposome-bound RecA floats to near

371

the top of the gradient, while unbound protein remains at the bottom or distributed across the

372

gradient. The fractions near the top of the gradient were analysed by SDS-PAGE and

373

visualized by silver staining method. The co-flotation capacity of RecA proteins was found to

374

be dependent on the type of phospholipid and the source of RecA. Figure 3A demonstrates

375

that EcRecA co-floated equally efficiently with liposomes containing PG, DOPE, PI:CL and

376

DOPE:CL. In contrast, co-flotation of EcRecA with liposomes containing PI was markedly

377

reduced, whereas it was abrogated with liposomes containing PS and PC. On the other hand,

378

mycobacterial RecA proteins were found to co-float equally efficiently with liposomes

379

containing PS, PC, PI, PG, DOPE and PI:CL (Figure 3B, C). Interestingly, while significant

380

amount of MtRecA was found to co-float with liposomes containing DOPE:CL, MsRecA co-

381

floated poorly. Taken together, the results from nitrocellulose strip and co-flotation assays

382

suggest that MsRecA has the capacity to directly interact with lipid vesicles containing PS

383

and PC. The lack of correlation between these two assays may be attributed to a subtle

384

difference in the amino acid sequence and/or composition and/or assay conditions.


Biochemistry 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 18 of 51 P a g e | 18

385 386

Figure 3. Interaction of RecA with phospholipids. (A-C) A co-flotation assay was performed

387

with RecA proteins from the indicated organism (E. coli, M. smegmatis or M. tuberculosis).

388

Lane 1, protein molecular weight markers (kDa), 2, control reaction performed in the absence

389

of liposomes, 3-9 reactions in the presence of 300 µM PS, PC, PI, PG, DOPE, PI:CL (70:30,

390

w/v) or DOPE:CL (70:30, w/v). The figure shown is a representative of three independent

391

experiments.

392

Quantitative analysis of RecA-phospholipid interaction by fluorescence measurements

393

The

fluorescence

properties

of

tryptophan

residues

are

sensitive

to

the

394

microenvironment, which has been widely used to gain insights into the interaction of

395

proteins and with bacterial membranes. To further validate the data from the binding assays,

396

the intrinsic fluorescence of Trp residues was used for studying RecA-phospholipid

397

interactions. To this end, a fixed concentration of RecA (0.5 µM) from the indicated bacterial

398

species was titrated against increasing concentrations of lipid vesicles containing different

399

phospholipids (5-125 µM). Figure 4A demonstrates that EcRecA bound to multiple acidic

400

phospholipids in a dose-dependent manner as assessed by changes in the fluorescence

401

emission spectra and an increase in fluorescence intensity. Consistent with the previous

402

studies,12 EcRecA bound with higher affinity to the lipid vesicles containing PI or a mixture


Page 19 of 51 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


403

of PI:CL; however, interactions with several phospholipids including PS, PC, PG, DOPE or a

404

mixture of PE:CL were also observed, albeit less efficiently.

405 406

Figure 4. The relative binding affinities of RecA proteins to different types of phospholipids.

407

The intrinsic fluorescence of RecA was measured in the absence and presence of increasing

408

concentrations of phospholipids (12.5, 25, 37.5, 50, 62.5, 75, 87.5, 100, 112.5 and 125 µM).

409

Panels A-C, the fluorescence intensity of EcRecA, MsRecA and MtRecA is plotted against

410

increasing amounts of liposomes. Table (D) shows the Kd values for the binding of EcRecA,

411

MsRecA and MtRecA to different phospholipids. N.D. denotes “not determined.” Error bars

412

represent the standard deviation of the mean of three independent measurements.

413

The binding of mycobacterial RecA proteins to lipid vesicles containing different anionic

414

phospholipids was measured in the same manner as that described for EcRecA (Figure 4B,

415

4C). Interestingly, MsRecA and MtRecA proteins exhibited slightly reduced binding affinity

416

for lipid vesicles containing PI or a mixture of PI:CL compared to EcRecA (compare Figure


Biochemistry 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 20 of 51 P a g e | 20

417

4A with Figure 4B, 4C). In comparison with the fluorescence changes observed upon binding

418

of EcRecA to liposomes, relatively higher lipid concentrations were required to obtain

419

changes in the fluorescence signal, thus indicating reduced affinity of MsRecA and MtRecA

420

proteins. In addition to the differences in Trp fluorescence intensity, sigmoidal dependence

421

was evident at the lower lipid concentrations.

422

To further characterize the affinity of RecA proteins for different types of anionic

423

phospholipids, saturation binding analyses were used to estimate the equilibrium dissociation

424

constants (Kd values). The data were fitted to a one site-specific binding model using the

425

equation Y = Bmax*X/(Kd + X). In this equation, Bmax represents the maximum specific

426

binding in the same units as Y (specific binding), Kd is the equilibrium binding constant, in

427

the same units as X (ligand concentration). Figure 4D summarizes the apparent Kd values of

428

EcRecA and mycobacterial RecA proteins to lipid vesicles containing different acidic

429

phospholipids. Notably, the E. coli and mycobacterial RecA proteins bind to each acidic

430

phospholipid tested with micromolar affinity; however, EcRecA exhibited slightly greater

431

affinity for lipid vesicles containing PI, PS or a mixture of PI:CL compared with its

432

homologues from mycobacteria. Taken together, these data suggest that although electrostatic

433

interactions have an important role in mediating the binding of RecA proteins to phospholipid

434

vesicles, subtle differences in the physico-chemical properties of protein and/or phospholipid

435

moieties may underlie the observed differences in binding affinities.

436

The stability of RecA nucleoprotein filaments was unaffected by phosphatidylinositol

437

It was previously demonstrated that the Z-DNA and ssDNA binding activity of EcRecA

438

was strongly inhibited by CL, PG, and phosphatidic acid.23 Further work showed that the

439

binding of EcRecA to ssDNA was unaffected by the presence of phospholipids.12 In view of

440

these conflicting findings, we studied the effects of phospholipids on the stability of


Page 21 of 51 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


441

preformed mycobacterial RecA nucleoprotein filaments. For this purpose, fluorescently

442

(FAM) labeled 50-mer ssDNA was incubated with varying concentrations of RecA (from the

443

indicated bacterial species) in the presence of ATPγS. The anisotropy changes were measured

444

and plotted against increasing concentrations of RecA (Figure 5A). The data were fitted to a

445

single site binding equation. The apparent Kd values for the RecA proteins were similar to

446

one another (Figure 5B). The Hill slope (h) value for all the three RecA proteins was >1,

447

suggesting multiple binding sites and positive cooperativity between the protomers of RecA.

448

In analogous experiments, the RecA nucleoprotein filaments formed with FAM labeled 50-

449

mer ssDNA were incubated with increasing concentrations of PI (as it displayed maximum

450

affinity with RecA proteins). We chose to use PI here as well as in other experiments

451

described below because it is concentrated on the inside leaflet of the lipid bilayer where it

452

plays crucial roles in cellular signalling, protein binding, and membrane dynamics.47

453

Interestingly, the stability of RecA-ssDNA filaments was found to be unaffected, indicating

454

that incubation with phosphatidylinositol does not dislodge RecA from ssDNA (Figure 5C).

455

Furthermore, the binding affinity (Kd) between RecA and ssDNA was unaffected by PI

456

(Figure 4D).


Biochemistry 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 22 of 51 P a g e | 22

457 458

Figure 5. Phosphatidylinositol does not affect the stability of RecA nucleoprotein filaments.

459

Panel (A): The changes in the anisotropy as a function of RecA proteins. The reaction

460

mixtures contained 50-mer ssDNA having 6-FAM at the 3′ end in the absence or presence of

461

(30, 60, 100, 160, 200, 300, 500 and 1000 nM) of EcRecA (red triangle), MsRecA (green

462

circle) or MtRecA (purple square). (B) Table shows the Kd values along with the Hill slope

463

and coefficient of determination (R2) values. (C) The change in the anisotropy of the RecA-

464

nucleoprotein filament in the presence of increasing concentrations of PI (25, 50, 100, 200,

465

500 and 1000 µM). The error bars represent standard deviation of the mean of three

466

independent measurements.

467

The ssDNA binding activity of mycobacterial RecA proteins was unaffected by

468

phospholipids

469

Since the results described in the previous section suggest that PI did not affect the

470

stability of RecA nucleoprotein filaments, we asked whether liposomes containing different


Page 23 of 51 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


471

phospholipids could impact the ability of RecA proteins to bind ssDNA. Towards this end,

472

32P-labelled

473

phospholipids in the assay buffer except RecA proteins for 5 min at 37 °C. The reaction was

474

initiated by the addition of either MsRecA or MtRecA and incubation was continued for

475

another 20 min. The reaction mixtures were analysed as described under Experimental

476

Procedures. The results show that increasing amounts of liposomes containing PI, a

477

combination of PI and CL or phosphatidylethanolamine and CL, exerted no significant

478

inhibition on the ssDNA binding activity of MsRecA or MtRecA as compared to the control

479

reaction (compare lane 3 with lanes 4-11, Figure 6A-F). The lack of inhibition is not

480

unexpected because the apparent equilibrium dissociation constants (Kd values) for the

481

binding of mycobacterial RecA with ssDNA are in the nM range (Figure 5B), while the Kd

482

values for RecA-phospholipid interaction are in the µM range, suggesting that RecA exhibits

483

high affinity towards ssDNA (Figure 4D).

50-mer ssDNA was preincubated with liposomes containing different types of

484 485

Figure 6. Phospholipids do not modulate the binding of RecA to ssDNA. The electrophoretic

486

mobility shift assay was performed as described under Experimental Procedures. The reaction

487

mixtures contained 32P-labeled ssDNA (83-mer), 1 µM MsRecA (A-C) or MtRecA (D-F) in

488

the absence or presence of 25-1000 µM of indicated liposomes. (A-F) Lane 1,


32P-labeled

Biochemistry 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 24 of 51 P a g e | 24

489

ssDNA, 2 ssDNA in the presence of indicated phospholipids; 3, complex of RecA with

490

ssDNA in the presence of ATP; 4-11, the RecA-ssDNA filament in the presence of 25, 50,

491

75, 100, 200, 400, 800 and 1000 µM of PI (A and D), PI:CL (B and E) and

492

phosphatidylethanolamine mixed with cardiolipin (C and F). The figure shown is a

493


494

Anionic phospholipids inhibit the ATPase activity of mycobacterial RecA proteins, but

495

ATP binding is unaffected

496

The generation of extended lengths of heteroduplex DNA and branch migration across

497

heterologous insertions during DNA strand exchange by RecA is tightly coupled to ATP

498

hydrolysis.1,7 The results presented above suggest that PI does not affect either the binding of

499

RecA to ssDNA or the stability of RecA nucleoprotein filaments (Figure 5 and Figure 6). So

500

the question remains: do anionic phospholipids play a role, if any, in regulating the ssDNA-

501

dependent ATPase activity of mycobacterial RecA proteins? In order to investigate this

502

question, MsRecA (or MtRecA) and liposomes containing PI or PI:CL were co-incubated

503

separately with ssDNA prior to the addition of ATP. The reaction mixtures were analysed as

504

described under Experimental Procedures. Interestingly, these experiments revealed that PI

505

inhibited ssDNA-dependent ATPase activity of MsRecA as well as MtRecA in a

506

concentration dependent manner (Figure 7A, 7E). In analogous experiments, the extent to

507

which the presence of PI:CL influenced ATP hydrolysis catalysed by mycobacterial RecA

508

proteins was determined. These experiments revealed that PI:CL inhibited the reaction at

509

concentrations that are comparatively greater than PI alone (7C, 7G).

510


Page 25 of 51 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


511

512 513

Figure 7. Phosphatidylinositol inhibits the ATPase activity of RecA proteins. Panels A-D

514

show the effect of PI:CL (70:30) on ATP hydrolysis catalyzed by MsRecA in the absence (A

515

and C) or presence (B and D) of SSB. Panels (E-H) show the effect of phospholipids on ATP

516

hydrolysis catalyzed by MtRecA. Panels A-H: lane 1, control reaction in the absence of

517

RecA; 2, ATP hydrolysis catalysed by RecA (MsRecA or MtRecA as indicated on top of the

518

panel); 3-10, reaction performed in the presence of 10, 20, 30, 40, 50, 60, 80 and 100 µM of

519

the indicated phospholipid. Panels I and J, the signal corresponding to 32Pi in panels A-H was

520

quantified and is shown graphically as a % inhibition of ATP hydrolysis catalysed by

521

MsRecA and MtRecA respectively, as a function of increasing concentrations of liposomes.

522

The error bars represent the standard deviation of the mean of three independent

523

measurements.

524

Previous work has demonstrated that RecA nucleoprotein filaments formed in the

525

presence of SSB are more stable, and remain active and extended relative to those formed


Biochemistry 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 26 of 51 P a g e | 26

526

with RecA alone.48,49 To explore whether SSB is capable of regulating the ATPase activity of

527

RecA in the presence of anionic phospholipids, it was pre-incubated with RecA, PI (or

528

PI:CL) and ssDNA prior to the addition of ATP. As expected, a side-by-side comparison of

529

ATP hydrolysis catalyzed by MtRecA or MsRecA in the presence of SSB revealed that the

530

inhibition was ~2-fold higher than that seen in reactions performed with RecA alone (Figure

531

7B, 7F and 7D, 7H). An explanation of these results is that SSB facilitates the binding of

532

RecA onto ssDNA as well as provides stability to the RecA nucleoprotein filament. The

533

quantification of results from these experiments revealed that the degree of inhibition was

534

similar for both MsRecA and MtRecA in the presence or absence of SSB (Figure 7I, 7J). To

535

allow comparison with published work, further experiments were performed to test the effect

536

of anionic phospholipids on ATP hydrolysis catalysed by EcRecA. In these experiments,

537

ATPase activity was measured in the presence of PG or PE:CL and with or without SSB.

538

Consistent with previous studies,12 the ATPase activity of EcRecA was inhibited in the

539

presence of PG or PE:CL (Figure 8). Taken together, these experiments demonstrate that the

540

interaction of liposomes containing anionic phospholipids with stable RecA nucleoprotein

541

filaments was enhanced, consequently leading to an increased inhibition of the ATPase

542

activity of mycobacterial RecA proteins.


Page 27 of 51 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


543 544

Figure 8. Phosphatidylglycerol and a mixture of phosphatidylethanolamine and CL inhibit

545

ATP hydrolysis activity of EcRecA. Panels A-D: lane 1, control reaction in the absence of

546

EcRecA; 2, ATP hydrolysis catalysed by EcRecA in the absence of any phospholipid; 3-10,

547

reactions performed in the presence of 10, 20, 30, 40, 50, 60, 80 and 100 µM of

548

phospholipids as indicated at the top of each panel. Panel E, the signal corresponding to 32Pi

549

in panels A-D was quantified and is shown graphically as a % inhibition of ATP hydrolysis

550

as a function of increasing concentrations of phospholipids.


Biochemistry 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 28 of 51 P a g e | 28

551 552

Figure 9. Anionic phospholipids do not affect the ability of RecA proteins to bind ATP.

553

Panels A-C: lane 1, MsRecA bound to [γ-32P]ATP in the absence of phospholipids. Lanes 2-

554

6, in the presence of 10, 25, 50, 75 and 100 µM of the indicated phospholipids. Panels D-F:

555

lane 1, MtRecA bound to [γ-32P]ATP in the absence of phospholipid. Lanes 2-6, in the

556

presence of 10, 25, 50, 75 and 100 µM of indicated phospholipid. (G) Lane 1, EcRecA bound

557

to [γ-32P]ATP; 2-6 in the presence of 10, 25, 50, 75 and 100 µM of PG. The figure shown is a

558

representative of three independent experiments. Panel H, % of ATP bound as a function of

559

increasing concentrations of phospholipids. The error bars represent the standard deviation of

560

the mean of 3 independent experiments.

561 562

It is possible, indeed likely, that the inhibition of ATPase activity in the presence of

563

anionic phospholipids may be attributable to the inability of RecA proteins to bind ATP. To

564

test this premise, EcRecA, MsRecA or MtRecA were incubated separately with [γ-32P]ATP

565

in the absence or presence of the indicated anionic phospholipid and then the reaction

566

mixtures were exposed to ultraviolet light at 254 nm. The products of the cross-linking assay

567

were analyzed by SDS-PAGE, and [γ-32P]ATP cross-linked to RecA was visualized using a

568

phosphorimager. A single protein band migrating at 38 kDa was seen representing the

569

covalently cross-linked RecA to radiolabeled ATP. Notably, there were no discernable

570

differences in the extent of the cross-linking of [γ-32P]ATP to EcRecA, MsRecA or MtRecA


Page 29 of 51 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


571

in the absence or presence of increasing concentrations of PI, PI:CL or PG (Figure 9A-G,

572

lanes 2-6). These results indicate that the contacts within the ATP-binding domain of RecA

573

are not impacted by the bound acidic phospholipids. Altogether, these results support the

574

notion that distinct mechanisms regulate ATP binding and its hydrolysis by RecA proteins.

575

Phospholipids do not affect the efficiency of DNA strand exchange promoted by RecA

576

proteins

577

In the next set of experiments, the effect of anionic phospholipids on RecA promoted

578

DNA strand exchange was examined using a three-strand exchange reaction. The 83-mer

579

ssDNA and homologous

580

absence or presence of increasing concentrations of anionic phospholipids (PI, PI:CL or

581

PE:CL). In this assay, a single reaction product is generated upon complete strand exchange:

582

32P-labeled

583

the reaction progressed to completion (>90%) and the presence of different anionic

584

phospholipids (as well as their increasing concentrations) had no effect on the DNA strand

585

exchange reaction promoted by all the three RecA proteins. Furthermore, in agreement with

586

previous studies with EcRecA,12 the inhibition of the ATPase activity of mycobacterial RecA

587

proteins by phospholipids had no significant effect on their DNA strand exchange activity.

588

However, these results are consistent with the model that ATPase activity is needed for the

589

disassociation of RecA monomers on ssDNA, but not for DNA strand exchange.1,7

32P-labelled

linear dsDNA (83 bp) were used as substrates in the

ssDNA displaced from 32P-labeled dsDNA. As shown in Figure 10 (panels A-I,)


Biochemistry 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 30 of 51 P a g e | 30

590 591

Figure 10. Phospholipids do not affect DNA strand exchange promoted by RecA proteins.

592

Panel A, Schematic diagram of the assay. Panels B-D show the effect of PI, PI:CL and

593

PE:CL on the strand exchange activity of MsRecA. Panels E-G, the effect of PI, PI:CL and

594

PE:CL on the strand exchange activity of MtRecA. Panels H-J, the effect of PI, PI:CL and

595

PE:CL on the strand exchange activity of EcRecA. Panels B-J: lane 1, control reaction in the

596

absence of RecA; 2, reaction promoted by RecA from the specified source, 3-11, reactions

597

catalysed by RecA in the presence of 25, 50, 75, 100, 125, 150, 175, 200 and 250 µM of the

598

indicated phospholipid. The band corresponding to the displaced ssDNA (product of strand


Page 31 of 51 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


599

exchange) in panels B-J was quantified and the data are shown graphically in panel K. The

600

error bars represent the standard deviation of the mean of three independent experiments.

601

Phosphatidylinositol inhibits the RecA co-protease activity of LexA repressor

602

The damage to DNA triggers RecA coprotease activity via activation of the SOS

603

response pathway. During normal growth conditions, approximately 40 unlinked SOS

604

response genes are repressed by the LexA repressor.1, 7 The co-protease activity of the LexA

605

repressor is catalyzed by the activated form of RecA through a nucleoprotein filament formed

606

on ssDNA at the site of DNA damage. A previous report has demonstrated that in E. coli by

607

inhibiting ATPase activity of RecA, thereby facilitating the stability of RecA nucleoprotein

608

filament, anionic phospholipids enhance the SOS response.12

609

possible role of anionic phospholipids on the RecA-induced SOS response in mycobacteria, a

610

co-protease assay was performed as described.41 First, the ability of MtRecA to induce the

611

auto-cleavage of MtLexA in the presence of increasing concentrations of PI was

612

characterized. In the absence of PI, MtRecA catalyzed the cleavage of MtLexA resulting in

613

the formation of two cleaved peptides/products (designated as CP1 and CP2). The addition of

614

increasing concentrations of PI markedly reduced the extent of cleavage of LexA, the highest

615

inhibition was seen at 500 µM (Figure 11A, D). We next studied the effect of PI on MsRecA

616

co-protease activity using MtLexA repressor. The cleavage products were analyzed and

617

quantified as described for the reaction catalysed by MtRecA. The quantified data showed

618

that MsRecA promoted the cleavage of MtLexA to a similar extent with equivalent

619

concentrations of reactants (Figure 11B, D). Since these results conflict with the

620

demonstrated inhibitory effect of CL on the auto-cleavage of LexA by EcRecA,12 the effect of

621

PI was tested under these conditions. The results revealed that PI inhibited the cleavage of

622

MtLexA by EcRecA at lower concentrations than that required for MtRecA under similar


To gain insights into the

Biochemistry 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 32 of 51 P a g e | 32

623

conditions (Figure 11C, D). Similar results were obtained in reactions containing a mixture of

624

PI and CL at a ratio of 70:30 (data not shown). Given the similar extent of cleavage promoted

625

by all three RecA proteins, these results allow us to suggest that the inhibition of co-protease

626

activity by PI and CL is not specific to mycobacterial RecA proteins, but rather that it is a

627

general phenomenon. Despite these similarities, one caveat is that the experiments with

628

EcRecA and MsRecA were conducted with the heterologous protein, LexA from M.

629

tuberculosis. However, we note that LexA is highly conserved across all families of bacteria

630

and shares a high degree of sequence similarity.50-52

631 632

Figure 11. Phosphatidylinositol suppresses RecA coprotease activity. The assay was

633

performed with MtRecA (A), MsRecA (B) or EcRecA (C) in the presence of increasing

634

concentrations of PI. Panels (A-C): lane 1, molecular mass markers (kDa); lane 2, MtRecA,

635

MsRecA or EcRecA; 3, MtLexA; 4-11, the indicated RecA and MtLexA were incubated with

636

50, 100, 150, 200, 300, 400 and 500 µM of PI. Panel D, the graph depicts the % of the

637

cleaved MtLexA in the presence of increasing concentrations of PI. The error bars represent

638

the standard deviation of the mean of three independent experiments.

639

Subcellular localization of RecA in SOS-induced and uninduced M. smegmatis cells


Page 33 of 51 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


640

In comparison with E. coli and B. subtillis RecA,11-14, 19-22 there is almost no data on the

641

properties of mycobacterial RecA proteins in live cells. To investigate the subcellular

642

distribution patterns of mycobacterial RecA, a recombinant plasmid having the M. smegmatis

643

recA gene fused in-frame to the gfp gene was constructed. However, the data from in vivo

644

localization of E. coli GFP-RecA fusion protein is complicated by the fact that fusion protein

645

yields loss-of-function phenotypes to RecA.13, 53 However, E. coli RecA fusion protein was

646

found localized outside of the nucleoid and associated with the inner membrane.12,13 In this

647

work, we first determined the ability of MsRecA-GFP fusion protein to complement the UV-

648

repair deficiencies of a M. smegmatis mc2155 recA strain. We observed partial

649

complementation of the UV sensitivity of M. smegamtis mc2155 recA strain by a plasmid-

650

borne gfp-recA translational fusion gene, indicating that MsRecA-GFP is partially active in

651

recombinational DNA repair (data not shown). These results are consistent with partial

652

complementation of UV-repair deficiencies of E. coli recA strain by recA-gfp translational

653

fusion gene.13, 53

654

The exponentially growing M. smegmatis mc2155 cells bearing recA-gfp recombinant

655

plasmids (expressing the MsRecA-GFP fusion protein) under normal conditions were imaged

656

by confocal microscopy. Similarly, cell imaging experiments were carried out with M.

657

smegmatis mc2155 cells (expressing the MsRecA-GFP fusion protein) exposed to 25 J/m2 UV

658

radiation at 254 nm. During its exponential growth, RecA fused GFP forms large and small

659

foci (in 10-15% of cells) that are distributed throughout the cell. After DNA damage, DNA

660

bound RecA-GFP foci increase 2 to 3-fold in the majority of cells (~80–90%), indicating that

661

DNA damage triggers the formation of new RecA foci that are localized at the middle of the

662

cell.12-14, 54


Biochemistry 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 34 of 51 P a g e | 34

663

The log phase M. smegmatis mc2155 cells grown under non-DNA-damaging conditions

664

(~25% of cells) displayed unipolar RecA-GFP foci, while ~75% of cells exhibited bipolar

665

RecA-GFP foci localized at the cellular poles, outside of the nucleoid and presumably

666

associated with anionic phospholipids in the inner membrane (Figure 12 A, panel i-iv). These

667

could be “storage structures” of RecA, although their function and significance in DNA

668

recombination/repair and SOS responses remain unclear. The RecA-GFP foci are neither

669

found dispersed in the cytoplasm nor are associated with DAPI staining, suggesting that the

670

RecA foci are occluded from the nucleoid during normal growth conditions (Figure 12 C, i).

671

Importantly, a change in the number and pattern of RecA-GFP foci as well as their

672

distribution was seen in M. smegmatis mc2155 cells after UV irradiation. Instead of being

673

localized to the cellular poles, cells contained four to six compact cluster(s) of discrete RecA-

674

GFP foci of various sizes within each cell (Figure 12 B, i-iv, C, ii and iii). Nevertheless,

675

RecA-GFP was not seen forming bundles or extended filaments of fluorescent signals

676

analogous to that seen in E. coli.11 However, GFP alone does not display polar localization in

677

M. smegmatis cells rather it was uniformly distributed throughout the cytoplasm highlighting

678

that the foci observed are indeed being the property of RecA (data not shown). One hallmark

679

of the SOS response in bacteria is the inhibition of cell division and the increase in cell

680

length. The length of M. smegmatis mc2155 cells was measured both before and after UV-

681

induced DNA damage. As expected, the average cell length was about 4.86 ±0.78 µm and 8.8

682

±1.95 m before and after UV irradiation, respectively. We conclude that there are two types

683

of foci in M. smegmatis mc2155 cells: one, DNA-less aggregates/storage structures associated

684

with the cell membrane and second, foci bound to DNA, possibly colocalized with the

685

replication/repair machinery.


Page 35 of 51 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


686 687

Figure 12. Subcellular localization of RecA in M. smegmatis. Microscopic analyses of

688

normal and UV-treated (25 J/m2) cells were performed as described under the Experimental

689

Procedures section. Fluorescent and bright field images were acquired with a confocal laser

690

scan microscope. Panel A (i-iv): normal growth conditions. Panel B (i-iv): images of the UV-

691

irradiated cells. Scale bars 10 µm. Panel C (i): enlarged image of single cell without UV.

692

Panel C (ii and iii): zoomed-in images of the cells after UV treatment. The figure shown is a

693


694

Elevated expression of recA results in increased RecA-membrane association

695

To verify the results from live-cell imaging experiments, the amount of RecA in the

696

cytoplasmic and membrane fractions of cells before and after UV-induced DNA damage was

697

analyzed. For this purpose, M. smegmatis mc2155 cultures grown for various periods of time

698

under normal growth conditions and after DNA damage (with mitomycin C) were used in the

699

preparation of cell membrane and cytoplasmic fractions. Equal amounts of protein from each

700

fraction was resolved by SDS-PAGE.42 The abundance of RecA in cytoplasmic and


Biochemistry 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 36 of 51 P a g e | 36

701

membrane fractions was assessed by Western blotting using antibodies against RecA, GroEL

702

and FtsH. Both GroEL and FtsH are good loading controls for the soluble and membrane-

703

bound protein fractions respectively. Consistent with previous studies,55, 56 an increase in the

704

levels of MsRecA was clearly immuno-detectable on the Western blots. In untreated cells,

705

RecA was nearly equally distributed between cytoplasmic and membrane-bound protein

706

fractions (Figure 13 A, C). Notably, the levels of RecA significantly increased in cells treated

707

with mitomycin C, indicating that the recA gene was activated during the SOS response. The

708

levels of RecA in the membrane protein fraction increased by 3 to 4-fold of the total protein

709

(Figure 13 B, D). However, RecA levels also increased by ~2-fold in the soluble cytoplasmic

710

fraction upon DNA damage (Figure 13B and C). Altogether, these data illustrate, for the first

711

time that significant amounts of RecA are associated with the cell membrane under normal

712

growth conditions and in SOS-induced M. smegmatis mc2155 cells.

713

714 715

Figure 13. Western blot analysis of RecA in the cytoplasmic and membrane fractions of

716

control and MMC-treated M. smegmatis mc2155 cells. Panels A and B show representative


Page 37 of 51 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


717

immunoblots of RecA, FtsH and GroEL protein levels at the indicated time intervals. The

718

intensity of signals in each fraction depicted in panels A and B was quantified and the data

719

are shown as histograms in panels C and D. The data are the mean of three independent

720

experiments, and the error bars represent the standard error.

721

DISCUSSION

722 723

Classically, the regulation of RecA protein function so far has focused on the formation

724

and/or activity of RecA nucleoprotein filaments by a variety of positive and negative

725

accessory protein factors.7 However, recent studies have revealed that the second messenger

726

cyclic di-AMP plays an important role in the negative regulation of mycobacterial RecA

727

proteins, but not E. coli RecA.57 To further inform our understanding of the regulation of

728

RecA function by endogenous small molecules, the potential roles of the plasma membrane

729

components was examined. We found that mycobacterial RecA proteins exhibit relatively

730

high affinity for CL and PI, the principal anionic constituents of the mycobacterial inner cell

731

membrane. The anionic character in CL (two phosphate groups) and PI (phosphatidic acid

732

backbone, linked via the phosphate group) may facilitate in vivo recruitment of RecA proteins

733

to the plasma membranes, albeit with different stoichiometric ratios. Most strikingly, anionic

734

phospholipids function as physiological regulators of RecA function: they impeded the DNA-

735

dependent ATPase activity of RecA proteins, while ATP-binding is not affected. Similarly,

736

the ability of RecA proteins to promote DNA strand exchange is not affected, but RecA-

737

stimulated autocatalytic cleavage of LexA repressor is inhibited. Moreover, MsRecA

738

physically associates with the plasma membrane, and live-cell imaging reveals that RecA foci

739

localize to the cell poles during normal metabolism and that these structures disassemble and

740

reassemble into several foci along the cell after DNA damage induction. Thus, these results

741

support the idea that the role of anionic phospholipids extends beyond that of being simple


Biochemistry 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 38 of 51 P a g e | 38

742

membrane anchoring devices for mycobacterial RecA proteins, to being regulators of their

743

function.

744

The patterns of binding of MsRecA and MtRecA to liposomes containing anionic

745

phospholipids were significantly different from those seen with EcRecA.12, 23 The data also

746

revealed that EcRecA binds weakly to PC, PI and PS compared to mycobacterial RecA

747

proteins. The PI and phosphatidylinositol mannosides which are rarely found in the

748

prokaryotic cell membrane are abundant in the inner membrane of the mycobacterial cell

749

envelope.28,58 The interaction between PI and mycobacterial RecA proteins not only enhances

750

the overall binding avidity but may also provide a degree of specificity toward the membrane.

751

However, interaction with PI alone may not be sufficient to anchor RecA onto the membrane;

752

therefore, specificity toward CL further ensures stable binding. By using anionic lipids with

753

different head groups, mycobacterial RecA proteins were found to have a high-affinity for PI,

754

while the effect is modest in the case of EcRecA.

755

It has previously been demonstrated that anionic phospholipids inhibit the ability of

756

EcRecA to bind ssDNA,23 implying that it cannot activate the SOS response pathway or

757

participate in recombinational DNA repair. One of the important findings of this study is that

758

both MsRecA and MtRecA can bind ssDNA and ATP as well as retain their capacity to

759

promote DNA strand exchange in the presence of acidic phospholipids. However, the

760

ssDNA-dependent ATPase activity of RecA proteins is markedly suppressed by anionic

761

phospholipids. The assay conditions used for the ATPase assay are not inhibitory, this is

762

demonstrated by the fact that under similar assay conditions, RecA proteins promote DNA

763

strand exchange. Hence, the mechanism underlying ATP hydrolysis catalyzed by RecA

764

proteins is seemingly independent of their binding to ssDNA and ATP. The finding that PI

765

and CL suppress the ATPase activity of MsRecA and MtRecA corroborate with EcRecA12


Page 39 of 51 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


766

and, consequently, PI and CL may provide a backup mechanism for the negative regulation

767

of the ATPase activity of RecA proteins. These differences imply that the amino acid

768

residues that are involved in the interaction between ionic lipids and ssDNA (and ATP) are

769

distinct and may require some specific features of MsRecA and MtRecA proteins.

770

Furthermore, it appears that anionic lipids may stabilize RecA bundles following DNA

771

damage by suppressing ATPase activity within RecA filaments. Taken together, these results

772

are consistent with the notion that ATPase activity is not essential for DNA strand exchange;

773

instead, ATP hydrolysis is required for the disassembly of RecA monomers in the

774

nucleoprotein filament.58-61

775

Other important findings reported in this study include the ability of PI, one of the three

776

primary integral phospholipids of the mycobacterial membrane, to abrogate RecA-stimulated

777

LexA cleavage, which is in contrast to E. coli RecA.12 This is further supported by the

778

finding that an increased amount of RecA is found in the membrane fraction of M. smegmatis

779

cells after DNA damage. Additionally, we observed a robust inhibition of mycobacterial

780

RecA catalysed ATP hydrolysis, albeit ATP binding was not affected. On the one hand, these

781

results reveal that the interaction between RecA and anionic phospholipids inhibits certain

782

specific functions; on the other hand, other functions are not affected.

783

explanation is that the dynamic behaviour of RecA is more complex and is regulated in

784

different ways, depending on the physiological state of the cell. Further studies are required

785

to test the above hypotheses and elucidate the mechanism by which RecA activity is

786

differentially regulated by anionic phospholipids.

One possible

787

The accumulating evidence strongly suggests that anionic phospholipids are important

788

regulators of DNA metabolism in cells. For example, anionic phospholipids (CL, PG), but

789

not neutral phospholipid (PE), inhibit the binding of E. coli and Staphylococcus aureus DnaA


Biochemistry 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 40 of 51 P a g e | 40

790

to oriC and decrease its affinity for adenine nucleotides.62-65 Furthermore, CL strongly

791

inhibits the reactions of all three eukaryotic DNA polymerases and also of terminal

792

deoxynucleotidyl transferase.66 To understand how acidic phospholipids regulate RecA

793

function, we need to understand how they control its affinity for phospholipids. Our

794

combined gel shift assay, fluorescence measurements and protein crosslinking assays show

795

that acidic phospholipids do not affect the binding affinity of RecA proteins to ssDNA or

796

ATP. In contrast, acidic phospholipids impede mycobacterial RecA ATPase activity

797

significantly. Similar observations have been made with EcRecA.12 It would be interesting to

798

identify the amino acid residues in RecA that interact with acidic phospholipids and inhibit its

799

ATPase activity. Additionally, PI inhibits RecA mediated LexA cleavage activity suggesting

800

that the interaction of RecA with acidic phospholipids of the membrane can abrogate

801

induction of the SOS response.

802

The high-affinity of mycobacterial RecA proteins for anionic phospholipids correlates

803

well with the data from Western blot analyses. Notably, increasing amounts of MsRecA were

804

found in the membrane fraction of living cells after DNA damage. The polar sequestration

805

suggests that the membrane might be used by RecA as a scaffold for a homology search and

806

the cytoplasmic pool of RecA might form the active repair centres on the damaged sites of

807

bacterial chromosome. In the context of living cells, RecA can undergo a phase transition to

808

form unipolar-to-bipolar foci and disassemble and reassemble into new foci in the cytosol

809

where it becomes available for DNA recombination/repair functions. These findings are

810

consistent with a model in which high concentrations of anionic phospholipids at cell poles

811

favours the increased localization of RecA at the cell poles.12-15 In summary, our results

812

suggest that anionic phospholipids play a significant role in regulating the function of

813

mycobacterial RecA proteins. Moreover, these studies provide fresh insights into the further


Page 41 of 51 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


814

characterization of the roles of anionic phospholipids in mycobacterial RecA protein

815

function.

816

We and others have previously proposed that RecA and LexA could serve as potential

817

therapeutic targets for augmenting the bacterial susceptibility to medically important

818

antimicrobials and combat the emergence of microbial resistance.41,67-69 While RecA is

819

homologous to Rad51, LexA has no obvious human homologues. Consequently, targeting the

820

SOS response by structure-specific agents against LexA cleavage seems a promising strategy

821

in the development of new therapies. Furthermore, these findings may have important

822

implications in therapeutic strategies for the reversion of drug resistance, bacterial fitness and

823

biofilm formation based on LexA cleavage. Although we have witnessed tremendous

824

progress in the broader understanding of RecA function, we still have much to learn about its

825

in vivo regulation and how its multifaceted activities are altered in physiological contexts,

826

especially in pathogenic organisms. To this end, regulation of the function of mycobacterial

827

RecA proteins by ionic phospholipids is a topic that awaits further investigation.

828

Funding sources

829

This work was supported by a grant (BT/CoE/34/SP15232/2015) under the Center of Excellence

830

from the Department of Biotechnology, New Delhi, to K. M, who is also the recipient of J. C. Bose

831

National Fellowship from the Department of Science and Technology and Bhatnagar Fellowship from

832

the Council of Scientific and Industrial Research, New Delhi

833

Conflict of interest: The authors declare that they have no conflicts of interest with the contents of

834

this article.

835

Notes: DP and KM conceived the study. DP performed the experimental work. KM and DP analyzed

836

the data. KM wrote the paper with DP. Both authors confirm that they have read and approved the

837

final manuscript.


Biochemistry 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 42 of 51 P a g e | 42

838

Abbreviations: BSA, bovine serum albumin; CL, cardiolipin; dsDNA, double-stranded DNA; DAPI,

839

4',6-diamidino-2-phenylindole; DTT, dithiothreitol; EcRecA, E. coli RecA protein; EDTA,

840

ethylene diamine tetraacetic acid; EMSA, electrophoretic mobility shift assay; GFP, green fluorescent

841

protein; HR, homologous recombination; IPTG, isopropyl β-D-1-thiogalactopyranoside; kb, kilobase;

842

PMSF, phenylmethylsulfonyl fluoride; ODN, oligonucleotide; MtLexA, M. tuberculosis LexA;

843

MsRecA, M. smegmatis RecA; MtRecA, M. tuberculosis RecA; PAGE, polyacrylamide gel

844

electrophoresis; PE, phosphatidylethanolamine; PI, phosphatidylinositol, PG,

845

phosphatidylglycerol; PVDF, polyvinylidene difluoride membrane; SDS, sodium dodecyl sulphate;

846

SSB, single-stranded DNA binding protein; ssDNA, single-stranded DNA.

847

Note Added in Proof – While this manuscript was under consideration, a study consistent

848

with a critical role of anionic phospholipids in the regulation of M. smegmatis RecA was

849

published (Wipperman, M. F., Heaton, B. E., Nautiyal, A., Adefisayo, O., Evans, H., Gupta,

850

R., van Ditmarsch, D., Soni, R., Hendrickson, R., Johnson, J., Krogan, N., and Glickman, M.

851

S. (2018) Mol. Cell 72,152-161).

852 853

REFERENCES

854

1.

Kowalczykowski, S. C., Dixon, D. A., Eggleston, A. K., Lauder, S. D., and Rehrauer,

855

W. M. (1994) Biochemistry of homologous recombination in Escherichia coli.

856

Microbiol. Rev. 58, 401-465.

857

2.

from RecA protein sequence comparisons. J. Bacteriol. 177, 6881-6893.

858 859

3.

862

Michel, B. (2005) After 30 years of study, the bacterial SOS response still surprises us. PLoS Biol. 3, e255. PMID: 16000023.

860 861

Karlin, S., Weinstock, G. M., and Brendel, V. (1995) Bacterial classifications derived

4.

Sassanfar, M., and Roberts, J. W. (1990) Nature of the SOS-inducing signal in Escherichia coli. The involvement of DNA replication. J. Mol. Biol. 212, 79-96.


Page 43 of 51 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


863

5.

Kreuzer, K. N. (2013) DNA damage responses in prokaryotes: regulating gene

864

expression, modulating growth patterns, and manipulating replication forks. Cold

865

Spring Harb. Perspect. Biol. 5, a012674. doi: 10.1101/cshperspect.a012674.

866

6.

Courcelle, J., Khodursky, A., Peter, B., Brown, P. O., and Hanawalt, P. C. (2001)

867

Comparative gene expression profiles following UV exposure in wild-type and SOS-

868

deficient Escherichia coli. Genetics 158, 41-64.

869

7.

Mol. Biol. 42, 41-63.

870 871

Cox, M. M. (2007) Regulation of bacterial RecA protein function. Crit. Rev. Biochem.

8.

Bell, J. C., Plank, J. L., Dombrowski, C. C., and Kowalczykowski, S. C. (2012) Direct

872

imaging of RecA nucleation and growth on single molecules of SSB-coated ssDNA.

873

Nature 491, 274-278.

874

9.

Flory, J., Tsang, S. S., and Muniyappa, K. (1984) Isolation and visualization of active

875

presynaptic filaments of RecA protein and single-stranded DNA. Proc. Natl. Acad.

876

Sci. U. S. A. 81, 7026-7030.

877

10.

recombination from the RecA-ssDNA/dsDNA structures. Nature 453, 489-484.

878 879

Chen, Z., Yang, H., and Pavletich, N. P. (2008) Mechanism of homologous

11.

Lesterlin, C., Ball, G., Schermelleh, L., and Sherratt, D. J. (2014) RecA bundles

880

mediate homology pairing between distant sisters during DNA break repair. Nature

881

506, 249-253.

882

12.

Rajendram, M., Zhang, L., Reynolds, B. J., Auer, G. K., Tuson, H. H., Ngo, K. V.,

883

Cox, M. M., Yethiraj, A., Cui, Q., and Weibel, D. B. (2015) Anionic phospholipids

884

stabilize RecA filament bundles in Escherichia coli. Mol. Cell 60, 374-384.

885

13.

Renzette, N., Gumlaw, N., Nordman, J. T., Krieger, M., Yeh, S. P., Long, E.,

886

Centore, R., Boonsombat, R., and Sandler, S. J. (2005) Localization of RecA in

887

Escherichia coli K-12 using RecA-GFP. Mol. Microbiol. 57, 1074-1085.


Biochemistry 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 44 of 51 P a g e | 44

888

14.

in RecA function in Escherichia coli. Mol. Microbiol. 67, 1347-1359.

889 890

Renzette, N., and Sandler, S. J. (2008) Requirements for ATP binding and hydrolysis

15.

Levin-Zaidman, S., Frenkiel-Krispin, D., Shimoni, E., Sabanay, I., Wolf, S. G., and

891

Minsky A. (2000) Ordered intracellular RecA-DNA assemblies: a potential site of in

892

vivo RecA-mediated activities. Proc. Natl. Acad. Sci. U. S. A. 97, 6791-6796.

893

16.

DNA double strand break repair centers in live cells. J. Cell Biol. 170, 357-366.

894 895

Kidane. D., and Graumann, P. L. (2005) Dynamic formation of RecA filaments at

17.

Badrinarayanan, A., Le, T. B., and Laub, M. T. (2015) Rapid pairing and

896

resegregation of distant homologous loci enables double-strand break repair in

897

bacteria. J. Cell Biol. 210, 385-400.

898

18.

repair of replication-dependent DNA breaks. J. Cell Biol. 217, 2299-2307.

899 900

Amarh, V., White, M. A., and Leach, D. R. F. (2018) Dynamics of RecA-mediated

19.

Garvey, N., St John, A. C., and Witkin, E. M. (1985) Evidence for RecA protein

901

association with the cell membrane and for changes in the levels of major outer

902

membrane proteins in SOS-induced Escherichia coli cells. J. Bacteriol. 163, 870-876.

903

20.

Inouye, M., and Pardee, A. B. (1970) Changes of membrane proteins and their

904

relation to deoxyribonucleic acid synthesis and cell division of Escherichia coli. J.

905

Biol. Chem. 245, 5813-5819.

906

21.

mutants of Escherichia coli. J. Mol. Biol. 104, 567-587.

907 908

22.

911

Gudas, L. J., and Pardee, A. B. (1976) DNA synthesis inhibition and the induction of protein X in Escherichia coli. J. Mol. Biol. 101, 459-477.

909 910

Gudas, L. J. (1976) The induction of protein X in DNA repair and cell division

23.

Krishna, P., and van de Sande, J. H. (1990) Interaction of RecA protein with acidic phospholipids inhibits DNA-binding activity of RecA. J. Bacteriol. 172, 6452-6458.


Page 45 of 51 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


912

24.

Zhang L, Rajendram, M., Weibel, D. B., Yethiraj, A., and Cui, Q. (2016) Ionic

913

hydrogen

914

insertion depth of RecA on multicomponent lipid bilayers. J. Phys. Chem. B. 120,

915

8424-8437.

916

25.

bonds and lipid packing defects determine the binding orientation and

Angala, S. K., Belardinelli, J. M., Huc-Claustre, E., Wheat, W. H., and Jackson, M.

917

(2014) The cell envelope glycoconjugates of Mycobacterium tuberculosis. Crit. Rev.

918

Biochem. Mol. Biol. 49, 361-399.

919

26.

mycobacterial cell wall. Annu. Rev. Microbiol. 69, 405-423.

920 921

Jankute, M., Cox, J. A., Harrison, J., and Besra, G. S. (2015) Assembly of the

27.

Lambert, P. A. (2002) Cellular impermeability and uptake of biocides and antibiotics

922

in Gram-positive bacteria and mycobacteria. J. Appl. Microbiol, 92, Suppl, 46S-54S.

923

PMID: 12000612.

924

28.

Bansal-Mutalik, R., and Nikaido, H. (2014) Mycobacterial outer membrane is a lipid

925

bilayer and the inner membrane is unusually rich in diacyl phosphatidylinositol

926

dimannosides. Proc. Natl. Acad .Sci. U. S. A. 111, 4958-4963.

927

29.

Biochem. 64, 29-63.

928 929

Brennan, P. J., and Nikaido, H. (1995) The envelope of mycobacteria. Annu. Rev.

30.

Liu, J., Takiff, H. E., and Nikaido, H. (1996) Active efflux of fluoroquinolones in

930

Mycobacterium smegmatis mediated by LfrA, a multidrug efflux pump. J. Bacteriol.

931

178, 3791-3795.

932

31.

biosynthesis, and beyond. Chem. Biol. 21, 67-85.

933 934 935

Marrakchi, H., Lanéelle, M.-A., and Daffé, M. (2014) Mycolic Acids: structures,

32.

Singh, P., Patil, K. N., Khanduja, J. S., Kumar, P. S., Williams, A., Rossi, F., Rizzi, M., Davis, E. O., and Muniyappa, K. (2010) Mycobacterium tuberculosis UvrD1 and


Biochemistry 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 46 of 51 P a g e | 46

936

UvrA proteins suppress DNA strand exchange promoted by cognate and noncognate

937

RecA proteins. Biochemistry 49, 4872-4883.

938

33.

Kumar, R. A., Vaze, M. B., Chandra, N. R., Vijayan, M., and Muniyappa, K. (1996)

939

Functional characterization of the precursor and spliced forms of RecA protein of

940

Mycobacterium tuberculosis. Biochemistry 35, 1793-1802.

941

34.

Snapper, S. B., Melton, R. E., Mustafa, S., Kieser, T., and Jacobs, W. R., Jr. (1990)

942

Isolation and characterization of efficient plasmid transformation mutants of

943

Mycobacterium smegmatis. Mol. Microbiol. 4, 1911-1919.

944

35.

465, 203-215.

945 946

36.

Sambrook, J., Maniatis, T., and Fritsch, E. F. (1989) Molecular cloning : a laboratory manual, 2nd ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.

947 948

Goude, R., and Parish, T. (2009) Electroporation of mycobacteria. Methods Mol. Biol.

37.

Georgalis, Y., Starikov, E. B., Hollenbach, B., Lurz, R., Scherzinger, E., Saenger, W.,

949

Lehrach, H., and Wanker, E. E. (1998) Huntingtin aggregation monitored by dynamic

950

light scattering. Proc. Natl. Acad. Sci. U. S. A. 95, 6118-6121.

951

38.

Arvind, T. A., and Rangarajan, P. N. (2016) Mouse Apolipoprotein L9 is a

952

phosphatidylethanolamine-binding protein. Biochem. Biophys. Res. Commun. 479,

953

636-642.

954

39.

Britt, R. L., Chitteni-Pattu, S., Page, A. N., and Cox, M. M. (2011) RecA K72R

955

filament formation defects reveal an oligomeric RecA species involved in filament

956

extension. J. Biol. Chem. 286, 7830-7840.

957

40.

Thakur, M., Kumar, M. B., and Muniyappa, K. (2016) Mycobacterium tuberculosis

958

UvrB is a robust DNA-stimulated ATPase that also possesses structure-specific ATP-

959

dependent DNA helicase activity. Biochemistry 55, 5865-5883.


Page 47 of 51 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


960

41.

Nautiyal, A., Patil, K. N., and Muniyappa, K. (2014) Suramin is a potent and selective

961

inhibitor of Mycobacterium tuberculosis RecA protein and the SOS response: RecA

962

as a potential target for antibacterial drug discovery. J. Antimicrob. Chemother. 69,

963

1834-1843.

964

42.

head of bacteriophage T4. Nature 227, 680-685.

965 966

43.

44.

Belisle, J. T., and Sonnenberg, M. G. (1998) Isolation of genomic DNA from mycobacteria. Methods Mol. Biol. 101, 31-44.

969 970

Parish, T., and Stoker N. G. (Eds.) (1998) Methods Mol. Med. Vol. 54, Mycobacteria protocols, Humana Press, Totowa, N.J., U.S.A. ISBN 978-1-59259-147-3.

967 968

Laemmli, U. K. (1970) Cleavage of structural proteins during the assembly of the

45.

Anilkumar, G., Srinivasan, R., and Ajitkumar, P. (2004) Genomic organization and in

971

vivo characterization of proteolytic activity of FtsH of Mycobacterium smegmatis

972

SN2. Microbiology 150, 2629-2639.

973

46.

Venkatesh, R., Ganesh, N., Guhan, N., Reddy, M. S., Chandrasekhar, T., and

974

Muniyappa, K. (2002) RecX protein abrogates ATP hydrolysis and strand exchange

975

promoted by RecA: insights into negative regulation of homologous recombination.

976

Proc. Natl. Acad. Sci. U. S. A. 99, 12091-12096.

977

47.

membrane dynamics. Nature 443, 651-657.

978 979

Di Paolo, G., and De Camilli, P. (2006) Phosphoinositides in cell regulation and

48.

Muniyappa, K., Shaner, S. L., Tsang, S. S., and Radding, C. M. (1984) Mechanism of

980

the concerted action of RecA protein and helix-destabilizing proteins in homologous

981

recombination. Proc. Natl. Acad. Sci. U. S. A. 81, 2757-2761.

982 983

49.

Kowalczykowski, S. C., Clow, J., Somani, R., and Varghese, A. (1987) Effects of the Escherichia coli SSB protein on the binding of Escherichia coli RecA protein to


Biochemistry 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 48 of 51 P a g e | 48

984

single-stranded DNA. Demonstration of competitive binding and the lack of a specific

985

protein-protein interaction. J. Mol. Biol. 193, 81-95.

986

50.

Luo, Y., Pfuetzner, R. A., Mosimann, S., Paetzel, M., Frey, E. A., Cherney, M., Kim,

987

B., Little, J. W., and Strynadka, N. C. (2001) Crystal structure of LexA: A

988

conformational switch for regulation of self-cleavage. Cell 106, 585−594.

989

51.

McDonald, J. P., Peat, T. S., Levine, A. S., and Woodgate, R. (1999) Intermolecular

990

cleavage by UmuD-like enzymes: Identification of residues required for cleavage and

991

substrate specificity. J. Mol. Biol. 285, 2199−2209.

992

52.

Mo, C. Y., Birdwell, L. D., and Kohli, R. M. (2014) Specificity determinants for

993

autoproteolysis of LexA, a key regulator of bacterial SOS mutagenesis. Biochemistry

994

53, 3158-3168.

995

53.

Handa, N., Amitani, I., Gumlaw, N., Sandler, S. J., and Kowalczykowski, S.C. (2009)

996

Single molecule analysis of a red fluorescent RecA protein reveals a defect in

997

nucleoprotein filament nucleation that relates to its reduced biological functions. J.

998

Biol. Chem. 284, 18664-186673.

999

54.

Irazoki, O., Aranda, J., Zimmermann, T., Campoy, S., and Jordi Barbé, J. (2016)

1000

Molecular interaction and cellular location of RecA and CheW proteins in Salmonella

1001

enterica during SOS response and their implication in swarming. Front. Microbiol. 7,

1002

1560. doi: 10.3389/fmicb.2016.01560

1003

55.

Durbach, S. I., Andersen, S. J., and Mizrahi, V. ( 1997 ). SOS induction in

1004

mycobacteria: analysis of the DNA-binding activity of a LexA-like repressor and its

1005

role in DNA damage induction of the recA gene from Mycobacterium smegmatis.

1006

Mol. Microbiol. 26, 643-653.

1007 1008

56.

Papavinasasundaram, K. G., Colston, M. J., and Davis, E. O. ( 1998 ). Construction and complementation of a recA deletion mutant of Mycobacterium smegmatis reveals


Page 49 of 51 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


1009

that the intein in Mycobacterium tuberculosis recA does not affect RecA function.

1010

Mol. Microbiol. 30, 525-534.

1011

57.

Manikandan, K., Prasad, D., Srivastava, A., Singh, N., Dabeer, S., Krishnan, A.,

1012

Muniyappa, K., and Sinha, K. M. (2018) The second messenger cyclic di-AMP

1013

negatively regulates the expression of Mycobacterium smegmatis recA and attenuates

1014

DNA strand exchange through binding to the C-terminal motif of mycobacterial RecA

1015

proteins. Mol. Microbiol. 109, 600-614.

1016

58.

Chiaradia, L., Lefebvre, C., Parra, J., Marcoux, J., Burlet-Schiltz, O., Etienne, G.,

1017

Tropis, M., and Daffé, M. (2017) Dissecting the mycobacterial cell envelope and

1018

defining the composition of the native mycomembrane. Sci. Rep. 7, 12807. doi:

1019

10.1038/s41598-017-12718-4.

1020

59.

Menetski, J. P., Bear, D. G., and Kowalczykowski, S. C. (1990) Stable DNA

1021

heteroduplex formation catalyzed by the Escherichia coli RecA protein in the absence

1022

of ATP hydrolysis. Proc. Natl. Acad. Sci. U. S. A. 87, 21-25.

1023

60.

Rehrauer, W. M., and Kowalczykowski, S. C. (1993) Alteration of the nucleoside

1024

triphosphate (NTP) catalytic domain within Escherichia coli RecA protein attenuates

1025

NTP hydrolysis but not joint molecule formation. J. Biol. Chem. 268, 1292-1297.

1026

61.

of ATP hydrolysis within a RecA filament. PLoS Biol. 3, e52. PMID: 15719060.

1027 1028

62.

Sekimizu, K., and Kornberg, A. (1988) Cardiolipin activation of DnaA protein, the initiation protein of replication in Escherichia coli. J. Biol. Chem. 263, 7131-7135.

1029 1030

Cox, J. M., Tsodikov, O. V., and Cox, M. M. (2005) Organized unidirectional waves

63.

Makise, M., Mima, S., Katsu, T., Tsuchiya, T., and Mizushima, T. (2002) Acidic

1031

phospholipids inhibit the DNA-binding activity of DnaA protein, the initiator of

1032

chromosomal DNA replication in Escherichia coli. Mol. Microbiol. 46, 245-256.


Biochemistry 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 50 of 51 P a g e | 50

1033

64.

Castuma, C. E., Crooke, E., and Kornberg, A. (1993) Fluid membranes with acidic

1034

domains activate DnaA, the initiator protein of replication in Escherichia coli. J. Biol.

1035

Chem. 268, 24665-24668.

1036

65.

Ichihashi, N., Kurokawa, K., Matsuo, M., Kaito, C., and Sekimizu, K. (2003)

1037

Inhibitory effects of basic or neutral phospholipid on acidic phospholipid-mediated

1038

dissociation of adenine nucleotide bound to DnaA protein, the initiator of

1039

chromosomal DNA replication. J. Biol. Chem. 278, 28778-28786.

1040

66.

polymerases with phospholipids. Biochim. Biophys. Acta 1007, 61-66.

1041 1042

Yoshida, S., Tamiya-Koizumi, K., and Kojima, K. (1989) Interaction of DNA

67.

Mo, C. Y., Culyba, M. J., Selwood, T., Kubiak, J. M., Hostetler, Z. M., Jurewicz, A.

1043

J., Keller, P. M., Pope, A. J., Quinn, A., Schneck, J., Widdowson, K. L., and Kohli, R.

1044

M. (2018) Inhibitors of LexA autoproteolysis and the bacterial SOS response

1045

discovered by an academic-industry partnership. ACS Infect Dis. 4, 349-359.

1046

68.

Alam, M. K., Alhhazmi, A., DeCoteau, J. F., Luo, Y., and Geyer, C. R. (2016)

1047

RecA inhibitors potentiate antibiotic activity and block evolution of antibiotic

1048

resistance. Cell Chem. Biol. 23, 381-391.

1049

69.

Bellio, P., Brisdelli, F., Perilli, M., Sabatini, A., Bottoni, C., Segatore, B., Setacci, D.,

1050

Amicosante, G., and Celenza, G. (2014). Curcumin inhibits the SOS response induced

1051

by levofloxacin in Escherichia coli. Phytomedicine 21, 430-434.

1052 1053 1054 1055 1056 1057


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The Anionic Phospholipids in the Plasma Membrane Play an

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