Mycobacterium tuberculosis UvrB Is a Robust DNA-Stimulated

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Mycobacterium tuberculosis UvrB is a robust DNA-stimulated ATPase that also possesses structure-specific ATP-dependent DNA helicase activity Manoj Thakur, Mohan B. J. Kumar, and Kalappa Muniyappa Biochemistry, Just Accepted Manuscript • DOI: 10.1021/acs.biochem.6b00558 • Publication Date (Web): 12 Sep 2016 Downloaded from http://pubs.acs.org on September 13, 2016

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Biochemistry 1

Mycobacterium tuberculosis UvrB is a robust DNA-stimulated ATPase that also possesses structure-specific ATP-dependent DNA helicase activity

Manoj Thakur, Mohan B. J. Kumar and Kalappa Muniyappa* Department of Biochemistry, Indian Institute of Science, Bangalore 560012, India

Running title: Mycobacterium tuberculosis UvrB is an ATP-dependent DNA helicase

*To whom correspondence may be addressed:

Tel.: +91 80 2293 2235/2360 0278 Fax: +91 80 2360 0814/0683 E-mail:

Funding Information This research was supported by a Senior Research Fellowship to M. T. (09/079(2548)/2012-EMR-I) from the Council of Scientific and Industrial Research, New Delhi, and a grant under the Center of Excellence (BT/CoE/34/SP15232/2015) from the Department of Biotechnology, New Delhi, to K. M. K. M. is the recipient of J. C. Bose National Fellowship (SR/S2/JCB-25/2005), Department of Science and Technology, New Delhi.

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Abbreviations: bp, base pair; BPB, bromophenol blue; BSA, bovine serum albumin; dsDNA, double-stranded DNA; DTT, dithiothreitol; EDTA, ethylene diamine tetraacetic acid; EMSA, electrophoretic mobility shift assay; EcUvrB, Escherichia coli UvrB; IPTG, isopropyl-1-thio-β-D galactopyranoside; MtUvrB, Mycobacterium tuberculosis UvrB; NER, nucleotide excision repair; nt, nucleotide; ODN, oligonucleotide; PAGE, polyacrylamide gel electrophoresis; SDS, sodium dodecyl sulfate; ssDNA, single-stranded DNA.


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Abstract Much is known about the Escherichia coli nucleotide excision repair (NER) pathway, however, very little is understood about the proteins involved, and the molecular mechanism of NER in mycobacteria. In this study, we show that Mycobacterium tuberculosis UvrB (MtUvrB), which exists in solution as a monomer, binds to DNA in a structure-dependent manner. A systematic examination of MtUvrB substrate specificity reveals that it associates preferentially with single-stranded DNA, duplexes with 3' or 5' overhangs and linear duplex DNA with splayed arms. Whereas E. coli UvrB (EcUvrB) binds weakly to undamaged DNA and has no ATPase activity, MtUvrB possesses intrinsic ATPase activity that is greatly stimulated by both single- and double-stranded DNA. Strikingly, we found that MtUvrB, but not EcUvrB, possesses the DNA unwinding activity characteristic of an ATP-dependent DNA helicase. The helicase activity of MtUvrB proceeds in the 3' to 5' direction and is strongly modulated by a non-translocating 5' singlestranded tail, indicating that in addition to the translocating strand it also interacts with the 5' end of the substrate. The fraction of DNA unwound by MtUvrB decreases significantly as the length of the duplex increases: it fails to unwind duplexes longer than 70 bp. These results, on the one hand, reveal significant mechanistic differences between MtUvrB and EcUvrB and, on the other, support an alternative role for UvrB in the processing of key DNA replication intermediates. Altogether, our findings provide insights into the catalytic functions of UvrB and lay the foundation for further understanding of the NER pathway in M. tuberculosis. Keywords: Nucleotide excision repair/UvrB/ATPase/DNA helicase/DNA replication


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Studies over the past four decades have uncovered distinct DNA repair pathways and myriad types of biochemical reactions, and have been recognized with Nobel Prizes for T. Lindahl, P. Modrich, and A. Sancar in 2015.1 Among the many forms of genomic damage, “bulky” DNA lesions that cause helical distortion in the double helix can lead to mutations and ultimately cancer.2-5 The bulky chemical adducts and inter-strand cross-links between adjacent base residues are induced by UV irradiation and chemical modification in all organisms.2-5 In all the three domains of life, various proteins/enzymes recognize DNA modifications and either repair them by direct reversal of the chemical alteration or target them for removal by a variety of different DNA repair pathways.2-5 One of the most studied and best understood DNA repair pathways is the nucleotide excision repair in Escherichia coli.1-3 The nucleotide excision repair (NER) pathway was reconstituted with highly purified proteins in the mid-1980s.1-3,6,7 Since that time a huge wealth of functional and structural knowledge has accumulated on the components of the NER pathway.8,9 The NER pathway in eubacteria and some archaea has the unique ability to remove a broad range of chemically and structurally unrelated DNA lesions including UV-induced photoproducts (cyclobutane dimers, 6-4 photoproducts, thymine glycol), bulky adducts, apurininc or apyrimidinic (AP) sites and cross-links, albeit with variable efficiencies.2,4,9 In E. coli, NER is a multistep, ATP-dependent process catalyzed by UvrABC endonuclease, encoded by uvrA, uvrB, and uvrC genes.10 UvrABC endonuclease, a heterotrimeric enzyme complex, recognizes the damage and excises a short oligonucleotide containing lesion by the successive action of its constituent subunits.2,4,9 The mechanism of NER is essentially conserved across evolution, although it is considerably more complex in eukaryotic cells than in bacteria.2,3,5 The general working model of E. coli NER envisages a symmetric


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complex of UvrA2B2 which scans DNA for the modified sites.2,4,8 Specifically, UvrA recognizes damage-induced structural distortions in the DNA,11-15 and then the damage is passed from the dimeric form of UvrA to UvrB, resulting in a tight UvrB–DNA pre-incision complex at the site of the damage.16-20 During these events, UvrB plays a central role in the functioning both in damage recognition/repair and by direct interaction with all the other components in the pathway: UvrA, UvrC, UvrD, and DNA pol I.21-25 Further, the UvrA2•B2 symmetric complex unwinds duplex DNA, engages UvrB at the damage site and facilitates UvrA eviction.17-19; 22-30 During the formation of the pre-incision complex, UvrB uses a βhairpin to verify the modified nucleotide and inserts itself between the two strands and clamps the substrate.31-33 Concomitant with the dissociation of UvrA from UvrB abetted by ATP hydrolysis,2,3,21 UvrC interacts with UvrB’s carboxy-terminal domain and cleaves the phosphodiester bonds, 8 nucleotides 5' and 4–5 nucleotides 3' to the modified site.34-38 The post-incision complex is displaced by the dual action of UvrD and DNA pol I, which remove the incised 12-mer.34-37 Finally, a DNA pol I fills the gap followed by ligation of DNA ends by LigA.6,7 Although UvrABC endonuclease-mediated excision repair in E. coli has been studied in considerable depth, these studies have left several questions unanswered.8 Little is known about the limited helicase activity of UvrB and its substrate specificity. Additionally, there exist significant differences in the NER pathway between E. coli and in other bacteria, and less is known especially in clinically relevant bacterial pathogens such as Mycobacterium tuberculosis. Furthermore, Pseudomonas putida possesses a second copy of UvrA protein, UvrA2 - a class II UvrA homologue, which lacks a UvrB-binding domain and is closely related to the UvrA2 homologues in Mycobacterial, Streptomyces and Deinococcus


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radiodurans genomes.39,40 Interestingly, NER plays a dual role in P. putida: besides functioning in the repair of modified DNA, NER is important in the generation of mutations.40 Together, these findings prompted us to ask to what extent the E. coli model is applicable to M. tuberculosis. Towards this end, work has begun to identify and characterize the components of the M. tuberculosis NER pathway.15,41 A functional NER pathway is required for full infectivity and survival of various pathogens. For example, the disruption of uvrA in Yersinia pseudotuberculosis, Borrelia burgdorferi and M. tuberculosis decreased their pathogenicity.42-44 The M. tuberculosis

∆uvrB strains were highly sensitive to UV irradiation in vitro and to reactive oxygen and reactive nitrogen intermediates in the cell, and were attenuated for infection in iNOS-wild type and iNOS-knockout (iNOS−/−) mice.15,44 Furthermore, patient isolates of extensively drug-resistant M. tuberculosis harbor a unique uvrBA582V mutation that corresponds to a region in E. coli UvrB involved in interaction with both UvrA and UvrC.45 These reports provided the impetus for examining the biochemical properties of M. tuberculosis UvrB (henceforth called MtUvrB). We found that MtUvrB exhibits robust DNA-stimulated ATPase activity and broad, overlapping DNA-binding specificities. In contrast to E. coli UvrB (henceforth referred to as EcUvrB), MtUvrB exists as a monomer in solution and unwinds a broad range of DNA substrates with a preference towards linear duplex with splayed arms and a 3′ flap substrate, in a 3'→5' direction. These results support a model in which MtUvrB associates preferentially with a 3' overhang and moves along ssDNA in a 3' → 5' direction consistent with the findings from Bacillus caldotenax UvrB complexed with 3' overhang DNA.46 These findings, on the one hand, reveal significant mechanistic differences between MtUvrB and EcUvrB and, on the other, support an alternative role for


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UvrB in the processing of key DNA replication/repair intermediates. Finally, our findings provide novel insights into the functional properties of M. tuberculosis UvrB and lay the foundation for further understanding of the NER pathway in this respiratory pathogen. Experimental procedures Bioinformatics analysis. Amino acid sequence of M. tuberculosis UvrB was acquired from the TubercuList web server (http://www.Pasteur.fr/Bio/TubercuList). The amino acid sequences of other M. tuberculosis species, B. subtilis, Thermotoga maritima, Thermus thermophilus, were also retrieved from Uniprot databases and analyzed for domain architecture and aligned using a multiple alignment algorithm with the Clustal series of programs and visualized using Jalview. The MtUvrB amino acid sequence was analysed for domain architecture using the Conserved Domain Database in NCBI (http://www.ncbi.nlm.nih.gov/Structure/cdd/wrpsb.cgi). Isolation, expression and purification of MtUvrB. The MTCY01B2.25 cosmid, which bears the M. tuberculosis uvrB gene, was obtained from Institut Pasteur, Paris, France. The nucleotide sequence of M. tuberculosis H37Rv (Rv1633) uvrB ORF was identified using the TubercuList database (http://www.Pasteur.fr/Bio/TubercuList). The coding sequence corresponding to the M. tuberculosis H37Rv uvrB gene was amplified via PCR from the cosmid MTCY01B2.25 using primers (forward primer, 5´GCGGTCGAGCATATGGTGCGCGCCGG-3´ and reverse primer, 5´CGCTCGCTGAAGCTTTCACTTCAGGCC-3´) carrying the restriction enzyme sites (underlined) for NdeI and HinD III. A single PCR cycle consisted of initial denaturation at 95 °C for 5 min, followed by 30 cycles of amplification with each cycle consisting of denaturation at 95 °C for 30 sec, then annealing at 62.3 °C for 45 sec and extension at 72 °C


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for 70 sec in the presence of 10% DMSO and 1 mM MgCl2 with Phusion high fidelity DNA polymerase (purchased from New England Biolabs). The PCR product was cloned directionally into the pET28a(+) (Novagen) expression vector, using terminal NdeI and HindIII restriction sites. The identity of the cloned M. tuberculosis uvrB gene was confirmed by sequencing. The recombinant plasmid designated pMtHis-uvrB. M. tuberculosis UvrB was over-expressed in the E. coli C41(DE3) strain harboring the plasmid pMtHis-uvrB. Bacteria were grown on 180 rpm in an LB broth, supplemented with 50 µg/ml kanamycin, at 37 °C to A600 nm = 0.5. Subsequently, the culture was incubated for 1 h at 4 °C and MtUvrB expression was initiated by the addition of IPTG to a final concentration of 0.1 mM, followed by a 12 h incubation at 18 °C. Cells were collected by centrifugation for 10 min at 6000 rpm at 4 °C and resuspended in buffer A (25 mM TrisHCl, pH 8, 400 mM NaCl, 2.5 mM β-mercaptoethanol and 20% glycerol). The resulting cell suspension was disrupted by sonication and the cell debris was removed by centrifugation in a Beckman Ti-45 rotor at 30000 rpm for 1 h at 4 °C. The supernatant was applied onto the Ni2+-NTA agarose column pre-equilibrated with buffer A. The column was washed with buffer B (25 mM Tris-HCl (pH 8.0), 1 M NaCl, 2.5 mM β-mercaptoethanol and 20% glycerol) until the wash buffer was devoid of A260 nm absorbing material. The column was then washed with 5 bed volumes of buffer C [25 mM Tris-HCl (pH 8), 50 mM NaCl, 2.5 mM β-mercaptoethanol and 20% glycerol] containing 2 mM imidazole. The bound proteins were eluted with linear gradient of imidazole (10 mM to 250 mM) in buffer C. Fractions containing MtUvrB were pooled and dialyzed against buffer C. The protein pool was diluted 10-fold with buffer C and applied to pre-equilibrated Q-Sepharose resin with buffer C. The column was then washed with 5 bed volumes of buffer C containing 100 mM NaCl.


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The bound proteins were eluted with a linear gradient of NaCl (200 mM to 500 mM) in 5 bed volumes of buffer C. The peak fractions were pooled and dialyzed against buffer C. MtUvrB was purified to >98% homogeneity (as assessed by SDS-PAGE).The purified protein, which was free of exo- or endonucleases and of endogenously bound nucleic acids, was stored at -80 °C. Site-directed mutagenesis of M. tuberculosis uvrB. A point mutation at the 146th position of the MtUvrB polypeptide from Cysteine to Alanine was generated by an overlapping PCR based strategy in the M. tuberculosis uvrB gene.47,48 First, two primers were designed incorporating the mutation from Cysteine to Alanine at residue 146. Vector pET28a(+) harboring the MtuvrB gene was used as a template and then two independent PCRs were performed. For the first set of PCR, the following primers were used: MtuvrB gene specific forward primer 5'-GCTGTCCATATGGTGCGCGCCGGCGGT-3' carrying an NdeI restriction enzyme site, and a mutant reverse primer 5'GGCCGTAGATGGCGGACACCGAA-3' carrying desired mutation. In the second set of PCR following set of primers were used: MtuvrB gene specific reverse primer 5'CTAATCAAGCTTTCACTTCAGGCCGGC-3' carrying HindIII restriction enzyme site and mutant forward primer 5'- TTCGGTGTCCGCCATCTACGGCC-3'. Both the PCR amplified products were gel purified, pooled together and used as templates for another round of PCR in which the full length product was amplified using the MtUvrB gene specific forward and reverse primers. Subsequently, the PCR product was cloned into the pET28a(+) expression vector, using terminal restriction enzyme sites and was named pMtUvrBC146A. The identity of MtUvrBC146A was confirmed by nucleotide sequencing.


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Expression and purification of EcUvrB. The pUNC211 plasmid, which bears the E. coli uvrB gene was a kind gift from Aziz Sancar. EcUvrB was over-expressed in the E. coli C41(DE3) strain harboring the plasmid pUNC211. E. coli cells were grown on 180 rpm in an LB broth, supplemented with 20 µg/ml tetracycline, at 37 ̊C to A600 nm = 0.5. Subsequently, the culture was incubated for 1 h at 4 °C and EcUvrB expression was initiated by the addition of IPTG to a final concentration of 0.1 mM, followed by another 12 h of incubation at 18 °C. Cells were collected by centrifugation for 10 min at 6000 rpm at 4 ̊C and resuspended in buffer A (25 mM Tris-HCl, pH 8, 50 mM NaCl, 2.5 mM βmercaptoethanol and 25% glycerol). Bacteria were then disrupted by sonication and the cell debris was removed by centrifugation in a Beckman Ti-45 rotor at 30000 rpm for 1 h at 4 °C. The supernatant was applied onto 10 ml of a Q Sepharose column pre-equilibrated with buffer A. The column was washed with 10 bed volume of buffer B (25 mM Tris-HCl (pH 8.0), 150 mM NaCl, 2.5 mM β-mercaptoethanol and 25% glycerol). The bound proteins were eluted with a linear gradient of NaCl (200 mM to 500 mM) in a buffer containing 25 mM Tris-HCl (pH 8.0), 2.5 mM β-mercaptoethanol and 25% glycerol. All the fractions were analysed on an SDS-polyacrylamide gel. The fractions containing the EcUvrB were pooled and precipitated with ammonium sulfate at 35 % saturation. The precipitated protein was collected by centrifugation at 18000 rpm at 4 °C for 1 h. The pellet was dissolved and dialyzed against buffer C (25 mM Tris-HCl (pH 8.0), 1 M NaCl, 2.5 mM β-mercaptoethanol and 10% glycerol). Dialyzed protein was applied onto 320 ml of a Superdex-200 column pre-equilibrated with buffer C. Following elution with buffer C, all the fractions were analyzed on SDS-PAGE and the fractions containing the protein of interest were pooled and dialyzed against buffer A. The dialyzed protein was then diluted 10-fold and applied onto


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10 ml of a Blue-Sepharose column pre-equilibrated with buffer A. The column was washed with 15 bed volumes of buffer B. The bound proteins were eluted with a linear gradient of NaCl (200 mM to 1 M) in a buffer containing 25 mM Tris-HCl (pH 8.0), 2.5 mM βmercaptoethanol and 25% glycerol. All the fractions were analyzed on SDS-PAGE and the fractions containing the protein of interest were pooled together and dialyzed against buffer A. EcUvrB protein was purified to near homogeneity as previously described.60 The purified proteins were stored at -80 ̊C. Immunoblotting analysis. Western Blots were prepared by electroblotting SDS-PAGE gels onto polyvinylidenedifluoride (PVDF) membranes and probing with anti-His antibody and anti-MtUvrB antibody as described.47 Prior to immunostaining, the membranes were blocked for 2 h at 25 °C with 5% skimmed milk in Tris-buffered saline [50 mM Tris-HCl (pH 8.0), 150 mM NaCl and 0.2% Tween-20]. The membranes were washed with Trisbuffered saline for 1 min and then incubated with primary antibody for 12 h at 4 °C (SigmaAldrich). Following washing the membranes with Tris-buffered saline 5 times, each for 15 min, they were incubated with horseradish peroxidase-conjugated anti-rabbit secondary antibody (Sigma-Aldrich) for 3 h at 4 °C. Finally, the blots were developed using Chemiluminescent substrates for horseradish peroxidase (Millipore) and imaged by ChemiDocImageQuant (GE LAS 4000). Construction of DNA substrates. Synthetic oligonucleotides used in this study are listed in Table S1, and DNA substrates in Table 1. The ODNs were radolabelled at the 5′ end by using [γ-32P]ATP and T4 polynucleotide kinase.47,48 Subsequently, DNA substrates were annealed by mixing appropriate combination of ODNs. For this purpose, stoichiometric amounts of purified ODNs were added to 100 µl of 0.3 M sodium citrate buffer (pH 7)


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containing 3 M NaCl. The ODNs were annealed by incubating at 95 °C for 5 min and then by slow cooling to 4 °C over a period of 2 h. The annealed substrates were gel purified by electrophoresis on a 10% (w/v) polyacrylamide gel in a 44.5 mM Tris-borate buffer (pH 8.3) containing 0.5 mM EDTA. The DNA substrates were excised from the gel, eluted into a TE buffer (10 mM Tris-HCl, pH 7.5, 1 mM EDTA) and precipitated with 0.3 M sodium acetate (pH 5.2) and 95% (v/v) ethanol. Chemical crosslinking of MtUvrB. Purified MtUvrB was treated with indicated amounts of freshly diluted glutaraldehyde. After incubation at 37 °C for 30 min, a 5x SDS sample loading buffer was added [10 mM Tris-HCl (pH 6.8), 40% glycerol, 12.5% SDS and 2.5 mM DTT containing 0.1% bromophenol blue] and incubation was extended for 5 min at 95 ºC. Samples were analyzed by 7.5% SDS–PAGE. The bands were visualized by staining with silver nitrate.47. Gel filtration chromatography. Gel filtration chromatography was performed using a Superose 12 10/300 GL column (GE Healthcare) on a Fast Performance Liquid Chromatography system with a 0.25 ml/min flow rate at 18 °C. The column was equilibrated with 25 mM Tris-HCl, 500 mM NaCl, pH 7.5, 1 mM EDTA, 5% glycerol and 2.5 mM DTT. A set of protein standards of known molecular mass such as vitamin B12 (1.3 kDa), myoglobin (17 kDa), ovalbumin (44 kDa), bovine γ-globulin (158 kDa and thyroglobulin (670 kDa) were used to construct the standard curve. A solution containing 1 mg/ml of MtUvrB was dialyzed against the buffer mentioned above and applied to the column. The column fractions were monitored for UV absorbance at 280 nm. The elution volume (Ve) corresponding to the protein peak was determined, and the approximate molecular weight deduced by interpolation on the standard curve.


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Static light scattering. Static light scattering experiments were performed using a Superose 6 10/300 column in a Wyatt TREOS Multi-Angle static Light Scattering instrument with an inline refractive index detector (Waters 2414 RI detector) and a multiwavelength UV detector (Shimadzu SPD-10A vp UV-Vis detector). Samples were centrifuged for 20 min at 13000 rpm before injection on a Superose 6 10/300 column (GE Healthcare). The column was pre-equilibrated with a buffer containing 20 mM Tris-HCl (pH 7.5), 350 mM NaCl, 2% glycerol and 2.5 mM DTT. The elution volume was plotted against the UV-signal and the molecular weight was derived from the light scattering data. Data analysis was carried out by using ASTRA (Wyatt Technology Corporation). Electrophoretic mobility shift assays. Electrophorectic mobility shift assays were carried out as previously described (15). Reaction mixtures contained 25 mM Tris-HCl (pH 7.5), 2.5 mM DTT, 5 mM MgCl2, 25 mM KCl, 0.25 mM ATP,32P-labelled substrate DNA, and indicated concentrations of MtUvrB. Reaction mixtures were incubated at 37 °C for 25 min and terminated by the addition of a loading dye (0.1% (w/v) bromophenol blue and xylene cyanol in 20% glycerol). In salt titration experiments, after incubating the reaction mixtures with MtUvrB at 37 °C for 20 min, the protein DNA complexes were challenged with the indicated concentrations of NaCl and incubation was continued for an additional 25 min at 37 °C. Samples were resolved by native PAGE in a 0.25X TBE buffer at 80 V at 4 °C. Gels were dried, and the bands were visualized using a Fuji FLA-9000 phosphorimager. The band intensities were quantified in UVItech gel documentation system using UVI-Band Map software (version 97.04) and plotted using GraphPad Prism (version 5.0). Fluorescence anisotropy. The assay was performed using a 50-mer ssDNA (ODN8, Table S1) containing 6-FAM at the 3' end, dsDNA (ODN7 annealed to ODN8; Table S1)


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containing 6-FAM at the 3' end of ODN8, and dsDNA (NDT annealed to ODN8; Table S1) containing 6-FAM at the 3' end of ODN8. Briefly, reaction mixtures (100 µl) contained 50 mM Tris-HCl (pH 7.5), 5 mM MgCl2, 5 nM DNA, 2.5 mM DTT, 5 mM ATP, 25 mM KCl, and increasing concentration of MtUvrB. After incubation at 37 °C for 30 min, fluorescence anisotropy was measured on a Beacon-2000 fluorescence polarization system (PanVera Corporation, Madison, WI) using excitation at 490 nm and emission at 520 nm at 22 °C. Anisotropy values were fit to a equation to calculate the Kd and h values. The equation used was Y=Bmax*X^h/( Kd^h + X^h). In this equation, Bmax corresponds to the maximum binding capacity, Kd is the apparent equilibrium dissociation constant and h is the Hill slope. ATPase assay. ATP hydrolysis was measured by thin layer chromatography on polyethyleneimine cellulose plates as previously described.15 Briefly, reaction mixtures (10 µl) contained 50 mM Tris-HCl (pH 7.5), 10 mM MgCl2, 200 nM DNA or no DNA, 1 mM DTT, 50 µg/ml BSA, 30 µM cold ATP, 200 pM of [γ-32P]ATP and indicated amount of MtUvrB. Reaction mixtures were pre-incubated at 37 °C for 5 min, prior to the addition of [γ-32P]ATP to start the reaction. After incubation for 60 min, reaction was terminated by the addition of EDTA to a final concentration of 15 mM, and 2 µl of aliquots were spotted on the TLC plates. The plates were developed in a solution containing 0.5 M LiCl, 1M HCOOH and 1 mM EDTA. The spots were visualized using Fuji FLA-9000 phosphorimager and quantified in UVItech gel documentation station using UVI-BandMap software (ver.97.04). The data were plotted using GraphPad Prism (ver.5.0). For kinetic parameters analysis, a constant amount of MtUvrB was incubated with increasing concentration of cold ATP spiked with radiolabeled [γ-32P]ATP as specified, in


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the absence or presence of DNA. For each concentration of ATP used, time course study was performed and slope was plotted using linear regression in GraphPad Prism to measure velocity. Kinetic parameters, Vmax and Km were determined by plotting velocity (1/v) versus substrate concentration (1/[S]) in double reciprocal Lineweaver–Burk plot. The turnover number (kcat) was calculated by ratio of Vmax to the total enzyme concentration used. Helicase assay. Reaction mixtures containing 25 mM Tris-HCl (pH 7.5), 1 mM MgCl2, 2.5 mM DTT, 0.5 nM of the indicated 32P-labelled substrate, 0.1 µg/ml BSA and indicated concentrations of MtUvrB were preincubated at 37 °C for 10 min as previously described.48 The reaction was started by the addition of 3 mM ATP and 5 nM unlabelled ODN, and incubation was continued for additional 90 min. In each experiment, a positive control for unwinding, prepared from incubation of the substrate at 95 °C for 5 min was included. Reactions were quenched with 1x quenching buffer (1 mg/ml proteinase K, 2.4 % (w/v) SDS, 100 mM EDTA, 0.12 % (w/v) bromophenol blue), and the deproteinized samples were resolved by non-denaturing 10% polyacrylamide gels (29:1 acrylamide/bisacrylamide) in a 0.5X TBE buffer [90 mM Tris-HCl, 89 mM boric acid, 1.98 mM EDTA (pH 8.3)] at 150 V. Gels were dried and the bands were visualized using the Fuji FLA-9000 phosphorimager. The band intensities were quantified in the UVItech gel documentation system using UVI-BandMap software (ver.97.04) and plotted using Graphpad Prism (ver.5.0). The percent of DNA substrate unwound was determined by the amount of 32Plabelled ODN released divided by the total amount of DNA substrate in the reaction. Biotin/streptavidin blocking assay. Biotin/streptavidin assay was performed as previously described.47 Briefly, the reaction mixture contained 25 mM Tris-HCl (pH 7.5), 1 mM MgCl2, 2.5 mM DTT, 0.1 µg/ml BSA, 0.5 nM substrate and 100 nM streptavidin. After pre-


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incubation for 10 min at 37 °C, MtUvrB was added in increasing concentrations together with 1 µM biotin (as a trap for free streptavidin) and incubation was extended for 5 min at 37 °C. The reaction was started with the addition of 3 mM ATP. After incubation for an additional 45 min, the reaction was quenched with a 1X quenching buffer (1 mg/ml proteinase K, 2.4% (w/v) SDS, 100 mM EDTA, 0.12 % (w/v) bromophenol blue). Samples were resolved by 10 % non-denaturing polyacrylamide gels (29:1 acrylamide/bisacrylamide) in a 0.5x TBE buffer (45 mM Tris-borate buffer (pH 8.3) containing 1 mM EDTA) at 150 V for 3 h at 4 °C. The gel was dried, exposed to a phosphorimaging screen and the images were analyzed using the Fuji FLA-9000 phosphorimager. The band intensities were quantified in a UVI-Tech gel documentation station using UVI-BandMap software (version 97.04) and plotted using GraphPad Prism (version 5.0). Results Comparison of E. coli and M. tuberculosis UvrB proteins. The full-length E. coli UvrB protein sequence was used as a query to search NCBI protein sequence databases for UvrB homologues in mycobacteria. We found potential uvrB homologues in the genomes of various mycobacterial species and in other bacterial species as well. The amino acid sequence of UvrB homologues appear to conform to the general structure and domain composition defined by the EcUvrB (Fig. S1). An analysis of the amino acid sequences of MtUvrB and EcUvrB revealed that they were nearly similar in length and share a 54% identity and 68.3% similarity, respectively (Table S2). Additionally, a strong correlation exists in the spatial arrangement of amino acid residues within the beta hairpin and helicase motifs of MtUvrB with that of EcUvrB, which couple the enzyme's ATPase and DNAbinding/unwinding functions. Like EcUvrB, MtUvrB contains 8 conserved motifs found


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Biochemistry 17

among the SF1 helicase superfamily members (Fig. S1 & S2A). The conserved domain analysis showed a domain-specific E-value of 1.14e-13 cd00046. We found a DEAD box motif at the N-terminal end that is unique to the helicase superfamily 2, a diverse family of proteins involved in ATP-dependent RNA or DNA unwinding reactions (Fig. S2B). Another specific hit, with an E-value of 2.96e-27/cd00079, suggested the relatedness of MtUvrB to the helicase superfamily c-terminal domain, found in DEXDc-, DEAD-, and DEAH-box proteins (Fig. S2B). Taken together, these features suggest that MtUvrB contains all of the structural properties of a DNA/RNA helicase necessary to couple ATP binding and hydrolysis to domain motion. Expression and purification of MtUvrB, EcUvrB and MtUvrBC146A. MtUvrB and EcUvrB were purified to homogeneity as described under “Experimental procedures.” These preparations showed a single band by SDS-PAGE and was judged to be >97% pure (Fig. 1A and 1C). The mutant variant, MtUvrBC146A, was expressed and purified in a manner similar to that of wild-type MtUvrB (Fig. 1B). The purity of the protein was ascertained by silver staining of proteins in the SDS/polyacrylamide gels, and their identity was further confirmed by Western blotting using either anti-His antibody or anti-MtUvrB antibody. Behavior of MtUvrB in aqueous solutions. There has been an ongoing debate about the oligomeric state of UvrB in solution. Gel filtration and sedimentation studies showed that EcUvrB exists in solution as a monomer.11 However, combined results from solution and Xray crystallographic studies suggest that UvrB from different organisms or the C-terminal domain of EcUvrB exist as homodimers.49-54 We employed three independent approaches to characterize the solution behaviour of UvrB. First, we used size-exclusion chromatography


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under non-denaturing conditions (Fig. 2A). MtUvrB consistently eluted between ovalbumin (44 kDa) and γ-globulin (158 kDa) with an approximate molecular weight of 77 kDa, in agreement with the expected size of the 78 kDa monomer. Under these conditions, the MtUvrB homodimer is expected to elute coinciding with the elution position of γ-globulin. To validate this observation (Fig. 2A), we next treated MtUvrB with glutaraldehyde, which crosslink’s lysine residues between interacting proteins. Incubation with increasing concentrations of glutraldehyde and then followed by SDS-PAGE analysis showed that MtUvrB migrated as a monomer (Fig. 2B). Further, size exclusion chromatography with multi-angle light scattering (SEC-MALS) analysis yielded a single peak of molecular mass 76 kDa, consistent with the theoretical mass (78 kDa) of the MtUvrB monomer (Fig. 2C). These results indicate that MtUvrB exists as a monomer in aqueous solutions in vitro. Characterization of the DNA substrate specificity of MtUvrB. E. coli UvrAB can specifically recognize damage in the DNA when it is localized at the end of a doublestranded fragment or in the unpaired region of a bubble substrate.26,28,55 These findings raise the question as to whether there exist preferred substrates for UvrAB because scanning complex might encounter various structurally distinct DNA substrates generated during DNA damage repair. Additionally, despite a central role for UvrB in excision, little is known about its substrate specificity other than that it binds modified ssDNA with high affinity in the absence of UvrA. To gain an insight into substrate specificity, we systematically analyzed the ability of MtUvrB to interact with a variety of DNA substrates using T-fluorescein adducted DNA substrates (i.e., single- and duplex DNA containing an internal fluorescein-dT adduct) as reference. All the assays were performed with 0.5 nM of the indicated 32P-labelled DNA (labelled at their 5' end) with increasing concentrations of


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Biochemistry 19

MtUvrB in the presence of 0.25 mM ATP and 5 mM MgCl2. The reaction products were analyzed by non-denaturing PAGE (EMSA) as described under “Experimental Procedures.” Under these conditions, the incubation of MtUvrB with fluorescein-ssDNA led to maximal binding within 2 min at 37 °C. An analysis of this interaction further revealed that MtUvrB bound to 12 structurally different DNA substrates in the following hierarchical manner: fluorescein-ssDNA (substrate 1, Table 1) > unmodified ssDNA (substrate 3) > linear duplex with splayed arms (substrate 6) > duplex with a 3'-flap (substrate 10) > duplex with a 3'overhang (substrate 8) > duplex with a 5'-overhang (substrate 7) > duplex with a 5'-flap (substrate 9) > replication fork (substrate 11) > bubble containing dsDNA (substrate 12) > blunt-ended unmodified dsDNA (substrate 4) > fluorescein-dsDNA (substrate 2) > Holliday junction (substrate 5) (Fig. 3A-M). However, in contrast to E. coli UvrB,56 MtUvrB bound weakly to bubble substrate (bubble size of 26 nt). Like E. coli UvrB which failed to bind dsDNA containing a lesion,56 MtUvrB binding to fluorescein-dsDNA and unmodified DNA was detectable, albeit remained low even at the highest MtUvrB concentrations tested. The lack of appreciable binding of MtUvrB to dsDNA is probably because MtUvrB dissociates very quickly from dsDNA. The specificity of complex formation was ascertained by competition experiments using an excess of unlabelled DNA. The N-terminal His6-tag did not influence DNA binding because its removal did not affect the affinity of MtUvrB for DNA. Although gel mobility assays are often used to determine the affinity and specificity of proteins in vitro to RNA and DNA, one limitation of the assay is that the products of the reaction are not at equilibrium during electrophoresis. We therefore measured the DNA binding activity of MtUvrB by fluorescence anisotropy. Toward this end, we monitored the


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binding of MtUvrB to fluorescein-dsDNA (substrate 2), and unmodified single- and doublestranded DNA (substrates 3 and 4) under EMSA conditions. For ssDNA, fluorescence anisotropy increased linearly as increasing amounts of MtUvrB were added to the reaction mixture, indicating multiple ssDNA-binding sites (Fig. S3A). On the other hand, with fluorescein-dsDNA fluorescence anisotropy increased at the beginning but with subsequent additions anisotropy changes became smaller and reached a plateau. The binding data were subjected to Hill binding model, which yielded Kd values and Hill coefficients that can be found in Fig. S3B. Intriguingly, MtUvrB binds with ~7-fold higher affinity to fluoresceindsDNA compared to ssDNA. Furthermore, this analysis yielded Kd values of 81.51±0.03 and 386±0.04 for fluorescein-dsDNA and unmodified dsDNA, respectively. Thus, about 5fold difference in Kd values between fluorescein-dsDNA and unmodified dsDNA support the idea that MtUvrB binds with higher affinity to the lesion containing dsDNA. However, the differences in Kd values between EMSA (see below) and fluorescence anisotropy indicate that the interaction of MtUvrB with DNA is more complicated and requires further study. Quantitative analysis of the binding of MtUvrB to DNA. The apparent equilibrium dissociation constant (Kd) of MtUvrB for ssDNA was ~2 to 4-fold lower compared to linear duplex with splayed arms, duplexes with 3' or 5' ssDNA tails and a 3' flap but not a 5' flap and a replication fork (Table 2). A comparison of Kd values revealed that MtUvrB exhibits ~30-fold higher binding affinity to fluorescein-ssDNA than E. coli or Thermus thermophilus UvrB.55-57 The Kd values for the replication fork, bubble substrate (bubble size of 26 nt) and a 5' flap substrate was in the µM range. Under these conditions, MtUvrB showed the


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Biochemistry 21

weakest affinity towards modified and unmodified duplex DNA as well as the Holliday junction, and, therefore it was not possible to calculate the Kd values. Binding of MtUvrB to modified ssDNA produces a salt stable complex. To determine the thermodynamic forces involved in the MtUvrB-DNA interaction, we examined the sensitivity of the complexes formed by MtUvrB with ssDNA to NaCl. After the formation of the MtUvrB-ssDNA complexes, they were challenged with increasing concentrations of NaCl. The samples were analyzed as described under “Experimental procedures.” We found that the complexes formed between MtUvrB and fluorescein-ssDNA (substrate 1) was very stable, even 1.6 M NaCl was not sufficient to dissociate the complexes. In contrast, >50% complexes formed between MtUvrB and unmodified ssDNA (substrate 3) were dissociated in the presence of 600 mM NaCl (Fig. S4). These results suggest that MtUvrB forms thermodynamically stable complexes with modified ssDNA compared to unmodified ssDNA, and that hydrophobic interactions are important for its binding to DNA. MtUvrB possesses ATPase activity, which is stimulated by DNA. The E. coli UvrA2●B2 symmetric complex uses the energy of ATP hydrolysis to track along unmodified DNA to locate a lesion, and DNA binding stimulates its ATPase activity.17,57,58 Previous studies have also shown that E. coli UvrB, by itself, has no measurable ATPase activity,59 but contains cryptic ATPase activity that is activated in the presence of UvrA and modified DNA.59-61 The cryptic ATPase activity arises from partially proteolyzed UvrB (UvrB*) as a minor contaminant in UvrB preparations, and this activity manifest only in the presence of high concentrations of salt and ssDNA.59,62 On the other hand, T. thermophilus UvrB exhibits intrinsic ATPase activity, which is stimulated by ssDNA.63 These conflicting reports prompted us to examine the ATPase activity of MtUvrB using [γ-32P]ATP as the


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substrate in the absence or presence of DNA. The reaction mixtures of ATPase assay were separated as described under “Experimental procedures.” As shown in Fig. 4A, MtUvrB by itself showed significant ATPase activity, and the extent of ATP hydrolysis increased in a concentration dependent manner. Interestingly, this activity was stimulated by both fluorescein-ssDNA (substrate 1) and fluorescein-dsDNA (substrate 2) to a similar extent of ~3-fold (Fig. 4B-D). In related experiments, we examined the time course of ATP hydrolysis in the absence and presence of DNA cofactors. The results revealed that the MtUvrB ATPase activity increased linearly up to 45 min before plateauing at around 1 h, and ~70% of ATP was hydrolyzed under these conditions (Fig. S5). However, the extent of stimulation was ~2 to 3-fold higher in the presence of modified DNA compared to unmodified DNA. Further, ATP hydrolysis plateau at 30% in the absence of DNA or in the presence of unmodified DNA, whereas it was 70% in the presence of fluorescein containing single- or double-stranded DNA. To determine the kinetic parameters, Km, Vmax and kcat, we measured MtUvrB ATPase activity at various concentrations of unlabelled ATP spiked with [γ-32P]ATP, in the absence or presence of fluorescein adducted ssDNA. Kinetic analysis of ATPase data shown in Fig. 5 suggests that the kcat value was ~2 times greater in the presence of fluorescein-ssDNA than in its absence. On the other hand, although the affinity of MtUvrB for ATP (Km) in the presence of fluorescein-ssDNA was quite similar with the affinity in its absence, the Vmax was enhanced by ~2-fold in the presence of ssDNA, indicating that the DNA cofactor increases the catalytic efficiency. Likewise, the kcat value for T. thermophilus UvrB in the presence of ssDNA was ~1.5 times greater than in its absence, whereas the Km was almost


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Biochemistry 23

same under both conditions.61 Since the E. coli UvrB was devoid of any detectable ATPase activity, we did not determine its Km and kcat values. MtUvrB exhibits robust helicase activity on structurally different DNA substrates. Previous studies have surmised that the E. coli UvrAB complex possesses DNAunwinding activity, which was restricted to the double-stranded regions of about 20 bp.57,64,65 Further, UvrB alone catalyzed the unwinding of partial dsDNA duplexes (17-mer ODN annealed to M13 ssDNA), albeit inefficiently.30,33,58 The robust MtUvrB ATPase activity in the presence of dsDNA, in contrast to the cryptic ATPase activity of E. coli UvrB, posits that MtUvrB might promote the unwinding of double-stranded DNA. To this end, we note that UvrB contains seven putative DNA helicase domains, a hallmark among members of the superfamily II DEAD-box helicases (Fig. S2; ref. 66). We therefore speculated that MtUvrB might exhibit DNA helicase activity similar to the canonical DNA helicases. To investigate the potential helicase activity of MtUvrB, assays were performed with different nucleotide cofactors and divalent metal ions using a 0.5 nM 32P-labelled linear duplex with splayed arms (substrate 6) as the substrate. The reaction products were analyzed by native PAGE as described under “Experimental procedures.” The optimal concentration of metal ions was determined by maintaining ATP at a fixed concentration (3 mM) and incubated with increasing concentrations of various divalent cations. Whereas Mg2+ and Mn2+ were the most effective cofactors, the other metal ions tested had little effect on MtUvrB helicase activity (Fig. S6, panels A-B). Both Mg2+ and Mn2+ were almost equally effective and elicited maximum activity between 1-2.5 mM (Fig. S6, panels C-E). Incubation with higher concentrations of Mg2+ or Mn2+ resulted in a progressive decrease in


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DNA unwinding activity possibly due to repulsive interactions. Similarly, the optimal concentration of nucleotide cofactors was determined by maintaining a fixed concentration of Mg2+ and titrating with various concentrations of nucleotide cofactors. Among the nucleotide cofactors tested, MtUvrB exhibited robust helicase activity in the presence of ATP and dATP with maximal unwinding over a broad range of concentrations, viz .,1-3 mM (Fig. S7B). However, ATPγS, AMP-PNP or other hydrolysable NTPs failed to support MtUvrB helicase activity. To gain further insights into the substrate specificity of MtUvrB helicase, we constructed 9 structurally different DNA substrates using appropriate ODNs listed in Table S1. To exclude any ambiguity in the interpretation of our results, all the substrates had a double-stranded region of 26 bp with 26 nt ssDNA tails/flaps/bubble/splayed arms or duplex regions. These include 26 bp duplex (HJ and RF, substrate 5 and 11, respectively), 26 bp duplex with 26 nt tails (3’ overhang and 5' overhang, substrate 8 and 7, respectively), 26 bp duplex with 26 nt flap (3' and 5' flap, substrate 10 and 9, respectively), 26 bp linear duplex with 26 nt splayed arms (substrate 6), 26 bp bubble flanked by 26 bp duplex (substrate 12) and 50 bp fluorescein-dsDNA (substrate 2). All the experiments were carried out in the presence of 1 mM Mg2+ and 3 mM ATP. The products were resolved by PAGE and visualized as described under “Experimental procedures.” Fig. 6, panels A-H, show the results of the helicase assay with increasing concentrations of MtUvrB. A quantitative analysis of the extent of product formation revealed significant differences among the substrates (Fig. 6J). MtUvrB exhibited robust helicase activity on linear duplex with splayed arms and a DNA substrate with a 3' flap. The replacement of the 3' flap substrate with a similar substrate containing a 5' flap or replication fork decreased unwinding to ~40%.


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Biochemistry 25

However, the fact that nearly all of the input splayed duplex and the 3' flap substrate was unwound suggests that MtUvrB acts catalytically, with a single enzyme unwinding several DNA molecules. A further analysis of MtUvrB helicase activity indicated that it displayed little or no activity on the Holliday junction, dsDNA with 3' or 5' overhangs, bubblecontaining and blunt-ended dsDNA. These results support the notion that MtUvrB requires a single-stranded region for unwinding and proceeds in a directional manner. The determination of the time courses of MtUvrB helicase activity with different DNA substrates suggested that the activity increased linearly and plateau around 60 min (see below). The efficiency of MtUvrB catalyzed unwinding declines proportional to the decrease in length of ssDNA tails. The foregoing studies show that MtUvrB catalyzed unwinding of splayed duplex containing a pair of 26 nt noncomplementary ssDNA tails was relatively more efficient and resulted in >95% strand displacement at the highest concentration tested. We next sought to determine the minimum length of noncomplementary ssDNA tail of a splayed duplex required for MtUvrB catalyzed unwinding. For this purpose, we used a set of splayed DNA substrates containing 26 nt (substrate 6), 18 nt (substrate 16), 10 nt (substrate 15) or 3 nt (substrate 14) noncomplementary ssDNA tails on both 3' and 5' ends of 26 bp duplex DNA and also a blunt-ended 26-bp dsDNA (substrate 13). The assays were performed using a fixed amount (0.5 nM) of the indicated 32P-labelled substrate with increasing concentrations of MtUvrB as described above. The results revealed that shortening of the ssDNA tail from 26 nt to 10 and 18 nt resulted in a gradual decease in the amount of DNA unwound and a minimum length of 3 nt at both the 3' and 5' ends was necessary for the MtUvrB catalyzed unwinding activity (Fig. 7A-E). No unwinding activity


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was observed with the blunt-ended 26-bp duplex DNA (Fig. 7E). Interestingly, the presence of a 26 nt ssDNA tail at the 5'-end had no effect on MtUvrB-catalyzed unwinding activity (Fig. 7F). We next sought to examine MtUvrB DNA helicase activity on substrates containing 3' and 5' flaps (substrate 10 and 9, respectively) in comparison with splayed duplex (substrate 6). The results from these assays showed that MtUvrB-catalyzed unwinding of a 26-bp DNA duplex with a 26-nt flap on 3' end was similar to that of the splayed duplex, with an appreciable strand displacement (>75%) during the 60-min time course (Fig. 8A-B). On the other hand, on using a similar DNA substrate with a 26 nt flap on the 5' end, we observed slightly lower levels of unwinding activity under similar conditions (Fig. 8C). Altogether, these results suggest that although the polarity of DNA unwinding by MtUvrB is likely to proceed in the 3' to 5' direction, it requires a forked duplex substrate with a 3 nt ssDNA tail at both 3' and 5' ends for optimal unwinding activity. The efficiency of MtUvrB catalyzed unwinding decreases with increasing length of dsDNA. We next investigated the length of dsDNA required for efficient unwinding by MtUvrB. Toward this end, we constructed a series of splayed DNA substrates having duplex regions of different lengths (varying from 20 to 90 bp, substrates 17-24, respectively) with all of them having 26 nt noncomplementary ssDNA tails. Assays were performed with a fixed amount (0.5 nM) of the indicated 32P-labelled substrate with increasing concentrations of MtUvrB as described above. We found that MtUvrB exhibited robust unwinding activity on substrates containing the 20 and 30-bp duplex region (Fig. 9AB, I). In fact, all of the input DNA substrate was unwound at ~70 nM of MtUvrB. In contrast, the efficiency of MtUvrB-catalyzed by DNA unwinding was progressively


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Biochemistry 27

decreased concomitant with an increase in the length of the duplex region from 40 to 80 bp (Fig. 9C-G, I). Thus, in the case of a substrate having 60 bp duplex region the unwinding efficiency was less than 50% (Fig. 9E). Further, no detectable unwinding activity was evident with the substrate containing the 90-bp duplex region at identical protein concentrations (Fig. 9H, I). MtUvrB unwinds DNA with a 3' to 5' polarity. Many DNA helicases translocate unidirectionally in a polar fashion abetted by NTP hydrolysis. Although the foregoing results imply that MtUvrB moves along ssDNA in a 3′ → 5′ direction, to ascertain these results, we used two forked DNA substrates with switched polarity.65,66 The unpaired ssDNA tails in such modified substrates have the same polarity: i.e., either 5′ or 3′ ends (substrate 25 and 26, respectively). We tested the ability of MtUvrB to unwind such modified substrates. Reactions were performed with a fixed amount (1 nM) of the indicated 32

P-labelled DNA substrate and varying concentrations (10 - 80 nM) of MtUvrB as

described above. Our results suggest that whereas ~75% of the substrate containing the unmodified 3′ end was unwound by MtUvrB (Fig. 10(b) and (d)), it failed to unwind substrates with modified 3′-ssDNA tails or modified on both 5′ or 3′ ends (substrate 27) (Fig. 10(A)(C) and (D)). To further confirm the polarity of DNA unwinding by MtUvrB, we used ssDNA substrates containing a biotin moiety conjugated to the nucleotide either at the 2nd or 4th position from the 3' or 5' end (substrate 29 and 28, respectively). One nM 32P-labelled ssDNA containing biotinylated nucleotide at the 3' or 5' end was incubated with 100 nM streptavidin in the helicase assay buffer at 37 °C for 10 min, followed with increasing concentrations of MtUvrB. The samples were processed as described above. The results of


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these experiments show that MtUvrB was able to displace streptavidin bound to ssDNA at the 5' end. In contrast, MtUvrB was unable to displace streptavidin from ssDNA containing the biotinylated nucleotide at the 3' end (Fig. 11, compare 11A with 11B). We note that the data obtained from streptavidin-blocking experiments is similar to those obtained with substrates having modified 3′ ends. Altogether, these results support the notion that streptavidin bound to the biotinylated nucleotide at the 3' end interferes with the translocation of the MtUvrB helicase; therefore, it translocates along ssDNA in a 3'-5' direction. Further, these results are consistent with those reported for B. caldotenax UvrB, which moves along ssDNA in a 3'-5' direction,46 and that nucleotides directly 3′ to the damage are buried within the EcUvrB molecule.69 Catalytic properties of E. coli UvrB and MtUvrBC146A. Given the findings above, which reveal that MtUvrB is a robust DNA-stimulated ATPase and exhibits strong helicase activity on replication associated DNA structures, we evaluated the substrate specificity as well as ATPase activities of EcUvrB. EMSA experiments were carried out using a set of DNA substrates (described above) and increasing concentrations of EcUvrB. Like MtUvrB, EcUvrB exhibited a somewhat greater affinity for a replication fork-like substrate than the lesion-containing ssDNA, and devoid of dsDNA binding activity (Fig. S78). Consistent with previous studies,2-4 EcUvrB showed higher binding affinity to bubble containing dsDNA than MtUvrB (compare Fig. 3K with Fig. S8H). Intriguingly, whereas MtUvrB failed to bind the Holliday junction, EcUvrB displayed robust binding activity (compare Fig. 3L with Fig. S8D). However, its functional significance remains to be investigated. We next investigated EcUvrB helicase activity on various substrates as a function of increasing enzyme concentrations. Whereas MtUvrB was able to unwind >80% of forked


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Biochemistry 29

duplex DNA in the reaction (Fig. 12A, lane 11), EcUvrB showed very weak helicase activity on the same substrate (Fig. 12A, lanes 3-10, Fig. 12E). However, EcUvrB generated the same radiolabeled product, albeit to substantially lesser extent.Under these conditions, like MtUvrB, EcUvrB catalyzed the unwinding of only a small fraction of the duplex substrate containing either 5' or 3' single-stranded arms (Fig. 12B-C). Further, EcUvrB displayed no intrinsic ATPase activity nor modified ssDNA was able to activate its ATPase activity (Fig. S9). Consistent with previous studies,30,33,57 we conclude that EcUvrB has exceedingly weak helicase activity. Based on the presence of conserved “helicase motifs”, putative helicases have been grouped into four superfamilies based on the extent of amino acid similarity and the organization of these conserved regions.70 Helicases of the superfamily 1 and 2 contain six conserved helicase motifs. In addition they also contain an additional conserved motif, called TxGx (Fig. S1), implicated in DNA binding and/or unwinding activity. The cysteine residue 146 in TxGx motif was replaced with alanine to assess whether the unwinding activity is intrinsic to MtUvrB. To this end, we first performed DNA binding, ATPase and unwinding activity of MtUvrBC146A as a function of increasing concentrations of the mutant enzyme. Whereas DNA binding and ATPase activities of MtUvrBC146A were comparable to those of wild-type MtUvrB (data not shown), we observed a ~4-fold reduction in the unwinding activity of MtUvrBC146A relative to wild-type MtUvrB (Fig.12D-E). The reduced unwinding activity of MtUvrBC146A strongly supports the idea that helicase activity is intrinsic to the wild-type MtUvrB polypeptide and that the cysteine residue in the TxGx motif contributes to helicase activity.


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Discussion Much is known about the NER pathway in E. coli;2-4 however, very little is understood about the proteins/enzymes involved, and the molecular mechanism of NER in mycobacteria. The prototype E. coli UvrB plays a central role in damage recognition, but its functional characteristics are currently not well understood. Amid our sparse knowledge about the properties of UvrB, we initiated biochemical and functional studies of M. tuberculosis UvrB. Notably, we found that MtUvrB possesses intrinsic DNA helicase and ATPase activities. Although the preferred substrates for UvrB within the bacterial cell remains unknown, the in vitro assays most often use a lesion containing ssDNA because, by itself, has no measurable affinity for damaged dsDNA.2-4 Single-stranded DNA, however, may not represent a likely target of UvrB under physiological conditions. To this end, using 12 structurally different DNA substrates, we show that MtUvrB associates preferentially with modified and unmodified ssDNA, linear duplex DNA with splayed arms and duplexes having 3' or 5' overhangs, indicating that MtUvrB can identify global conformational differences in its target DNA substrate(s). Although EMSA assays showed that MtUvrB lacks significant binding activity to modified dsDNA, fluorescence anisotropy measurements indicated that it exhibited higher affinity to modified dsDNA compared to ssDNA. Such variability is likely related to non-equilibrium conditions of the EMSA. Neverthelss, together with previous studies,15,41 the results presented here further our understanding of the NER pathway in M. tuberculosis. The findings from the E. coli paradigm have contributed significantly to the body of knowledge regarding NER pathway in eukaryotic organisms, however, several key questions about this pathway in E. coli have remained unanswered.8 Amongst these are (i)


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how does the UvrA2B (or UvrA2B2) complex discriminate between damaged and undamaged DNA; (ii) how is the damage detected among a wide array of structurally diverse lesions and substrates; (c) what is the function of individual proteins, their roles within the complex, and roles in alternative DNA-related processes such as replication. The latter possibility is consistent with the demonstration that E. coli uvrA and uvrB perform genetically separable functions.71,72 Other studies have found that DNA replication becomes dependent on uvrB in E. coli strains with a mutation affecting DNA pol I activity.73,74 Nevertheless, the molecular mechanism underlying its essential role in DNA replication is largely unknown. Our results corroborate the genetic studies and add further support to the notion that MtUvrB might play a critical role in DNA replication by the virtue of its ability to unwind linear duplexes with splayed arms and replication fork in normal or stressed cells. Three independent experimental approaches showed that MtUvrB exists in solution as a monomer. This result is consistent with results obtained for EcUvrB using similar methods.11 However, like EcUvrB, 18 it is possible that MtUvrB may dimerize while forming the pre-incision complex. Additionally, whereas T. thermophilus UvrB and B. caldotenax UvrB exist as monomers in solution,52,75 B. subtillis UvrB binds ssDNA as a dimer in the presence of a non-hydrolyzable ATP analog.53,76 As a growing number of DNA helicases have been characterized, a common property of most of the helicases in part is their ability to form defined oligomers in solution - homodimeric, heterodimeric or hexameric forms being the most common.77 Because MtUvrB exists as a monomer in solution, a pertinent question is whether MtUvrB can unwind the two complementary strands of dsDNA. Toward this end, one can envisage that MtUvrB dimerizes upon binding


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to DNA, and this may facilitate unwinding, as in the case of E. coli Rep and Bacillus stearothermophilus PcrA helicases.78,79 A part of the mechanism of excision of the damaged nucleotide by UvrABC endonuclease involves unwinding of dsDNA. However, the mechanism by which the UvrABC endonuclease complex unwinds dsDNA remains poorly understood. The wild-type EcUvrB exhibits cryptic ATPase activity,59,62 which can be unmasked by its cognate UvrA or upon removal of 40 amino acid residues from the C-terminus.59 MtUvrB contains amino acid sequence motifs that are common to many ATPases (Fig. S1). Interestingly, we found that modified single- and double-stranded DNA stimulated the ATPase activity of MtUvrB to similar maximal extents (~2-fold), as seen previously with T. thermophilus UvrB by ssDNA.63 Our finding that MtUvrB failed to form a stable complex with modified dsDNA, but stimulated ATPase activity was surprising. This implies that MtUvrB may associate with dsDNA, albeit transiently, and scans DNA for DNA damage in an ATP-dependent manner. The MtUvrBC146A mutant protein expressed and purified from E. coli C41(DE3) strain appears to be folded properly as as it binds ssDNA and exhibits DNA-stimulated ATPase activity, characteristics similar to that of wild-type MtUvrB, but is deficient in helicase activity. Although UvrB shares strong sequence similarities with helicases,66 little is known concerning its ability to unwind duplex DNA. It is thought that scanning for damaged DNA by the E. coli UvrA2B (or UvrA2B2) complex requires the ATPase and helicase activities of UvrB.30 EcUvrB alone failed to bind to any type of DNA.80 One model of damage recognition by the E. coli UvrA2B (or UvrA2B2) complex posits that upon binding at the damaged site, the “helicase activity” of UvrB unwinds DNA at the site by about 5 bp.3,4 The


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unwinding is facilitated by the wrapping of DNA around the UvrB protein and probably also by the damage itself, which is believed to destabilize the helix structure.53 The E. coli UvrA2B (or UvrA2B2) complex unwinds dsDNA from the 5' to 3' direction in a reaction dependent on ATP hydrolysis, needs at least 10 nt of ssDNA to initiate its unwinding activity, and is unable to proceed beyond 27 bp.31,58,64,65 Strikingly, we show here that MtUvrB has intrinsic ATP-dependent unwinding activity that is strongly influenced by the nature of the DNA substrate used. The fraction of DNA unwound decreases significantly as the length of the duplex increases, indicating a poor in vitro processivity. Overall, our results point out that UvrB’s ATPase activity couples with DNA unwinding activity. The ability of MtUvrB to unwind dsDNA is strongly modulated by the nature of the non-translocating 5'-ssDNA tail: at least 3 nucleotides at the 5'-tail are essential for MtUvrB unwinding activity. Although the mechanism by which a short 5' tail modulates the unwinding activity of MtUvrB is unknown, it is possible that a part of MtUvrB interacts with the 5' ssDNA tail at the junction making the enzyme competent for unwinding. We also observed a significant specificity of MtUvrB for the 3' flap over the 5' flap containing substrates. In addition to the robust unwinding of a linear duplex with splayed arms, MtUvrB also catalyzes the unwinding of duplex DNA with a 3' flap more efficiently compared to a similar substrate having a 5' flap. In contrast, the replication fork makes a modest but significant contribution to the unwinding activity of MtUvrB. Interestingly, however, MtUvrB failed to unwind duplex DNA with 3' overhangs, DNA with 5' or 3' overhangs, bubble-containing duplex DNA and the Holliday junction. We believe that the absence of MtUvrB helicase activity on blunt-ended dsDNA reflects a kinetic impediment; however, the mechanistic basis for its inability to unwind DNA with 3' or 5' overhangs


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needs further investigation. One possibility is that MtUvrB captures a small number of these structures as the dsDNA regions near the junction or fork undergoes “breathing” to expose ssDNA. Altogether, our studies are consistent with the notion that MtUvrB is a bonafide helicase. How do these results fit current mechanistic models of UvrB function during DNA damage recognition and incision? There are several notable differences as well as some interesting similarities between MtUvrB and EcUvrB. The similarities include that both associate preferentially with damaged ssDNA and show a very weak affinity towards dsDNA. The major difference that distinguishes EcUvrB from MtUvrB is that the former is devoid of ATPase and DNA helicase activities. As evidenced by our study with MtUvrB, and the one performed with T. thermophilus UvrB,63 these enzymes have intrinsic ATPase activity that is greatly stimulated by DNA. Together, these results point to the significant mechanistic differences in the NER machinery between E. coli and M. tuberculosis and suggest that there may be additional roles for the components of the NER pathway in mycobacteria. The latter seems more plausible because genetic studies have shown that in the absence of polymerase I, DNA replication becomes dependent on UvrB.71-73 Finally, our findings provide insights into the substrate specificity of M. tuberculosis UvrB and lay the foundation for further understanding of the NER pathway in this respiratory pathogen. Acknowledgements We grateful to Aziz Sancar and Nora Goosen for their generous gift of expression plasmids bearing the E. coli uvrB gene, and advice in the purification of EcUvrB. We gratefully acknowledge the anonymous reviewer for providing insightful comments, which


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improved the quality of the manuscript, and Niranjan V. Joshi for his helpful discussions on data analysis. Conflict of interest The authors declare that they have no conflicts of interest with the contents of this article. Author contributions M.T. and K. M. designed, analyzed, and interpreted the data. M.T. and M.B.J.K performed the experiments. K.M. wrote the paper with inputs from M.T. All authors reviewed the results and approved the manuscript. Supporting information Sequences of oligonucleotides used in this study (Table S1), comparison of the deduced amino acid sequences of E. coli UvrB with its homologues in various species of bacteria (Table S2), and Figures S1 to S8. This material is available free of charge via the Internet at http://pubs.acs.org. References 1. Friedberg, E.C. (2016) A history of the DNA repair and mutagenesis field: The discovery of base excision repair. DNA Repair (Amst) 37, A35-39. 2. Reardon, J.T., and Sancar, A. (2005) Nucleotide excision repair. Prog. Nucleic Acid Res. Mol. Biol. 79, 183-235. 3. Kisker, C., Kuper, J., and Van Houten, B. (2013) Prokaryotic nucleotide excision repair. Cold Spring Harb. Perspect. Biol. 5, a012591. 4. Truglio, J.J., Croteau, D.L., Van Houten, B., and Kisker, C. (2006) Prokaryotic nucleotide excision repair: the UvrABC system. Chem. Rev. 106, 233-252.


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29. Oh, E.Y., Claassen, L., Thiagalingam, S., Mazur, S., and Grossman, L. (1989) ATPase activity of the UvrA and UvrB protein complexes of the Escherichia coli UvrABC endonuclease. Nucleic Acids Res. 17, 4145–4159. 30. Moolenaar, G.F., Visse, R., Ortiz-Buysse, M., Goosen, N., and van de Putte, P. (1994) Helicase motifs V and VI of the Escherichia coli UvrB protein of the UvrABC endonuclease are essential for the formation of the preincision complex. J. Mol. Biol. 240, 294–307. 31. Moolenaar, G.F., Lotta Höglund, L., and Goosen, N. (2001) Clue to damage recognition by UvrB: residues in the β-hairpin structure prevent binding to non-damaged DNA. EMBO J. 20, 6140–6149. 32. Moolenaar, G.F., Monaco,V., van der Marel, G.A., van Boom, J.H., Visse, R., and Goosen, N. (2000) The effect of the DNA flanking the lesion on formation of the UvrB– DNA preincision complex. Mechanism for the UvrA-mediated loading of UvrB onto the damage. J. Biol. Chem. 275, 8038–8043. 33. Skorvaga, M., Theis, K., Mandavilli, B.S., Kisker, C., and Van Houten, B. (2002) The beta -hairpin motif of UvrB is essential for DNA binding, damage processing, and UvrC-mediated incisions. J. Biol. Chem. 277, 1553-1559. 34. Sancar, A., and Rupp, W. D. (1983) A novel repair enzyme: UvrABC excision nuclease of Escherichia coli cuts a DNA strand on both sides of the damaged region. Cell 33, 249–260. 35. Lin, J.J., and Sancar, A. (1992) Active site of (A)BC excinuclease. I. Evidence for 5’ incision by UvrC through a catalytic site involving Asp399, Asp438, Asp466, and His538 residues. J. Biol. Chem. 267, 17688–17692.


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36. Lin, J.J., Phillips, A.M., Hearst, J.E., and Sancar, A. (1992) Active site of (A)BC excinuclease. II. Binding, bending, and catalysis mutants of UvrB reveal a direct role in 3' and an indirect role in 5’ incision. J. Biol. Chem. 267, 17693–17700. 37. Verhoeven, E.E., van Kesteren, M., Moolenaar, G.F., Visse, R., and Goosen, N. (2000) Catalytic sites for 3' and 5' incision of Escherichia coli nucleotide excision repair are both located in UvrC. J. Biol. Chem. 275, 5120–5123. 38. Zou, Y., Walker, R., Bassett, H., Geacintov, N. E., and Van Houten, B. (1997) Formation of DNA repair intermediates and incision by the ATP-dependent UvrB-UvrC endonuclease. J. Biol. Chem. 272, 4820-4827. 39. Timmins, J., Gordon, E., Caria, S., Leonard, G., Acajjaoui, S., Kuo, M.-S., Monchois, V., and McSweeney, S. (2009) Structural and mutational analyses of Deinococcus radiodurans UvrA2 provide insight into DNA binding and damage recognition by UvrAs. Structure 17, 547–558 40. Tark, M., Tover, A., Koorits, L., Tegova, R., and Kivisaar, M. (2008) Dual role of NER in mutagenesis in Pseudomonas putida. DNA Repair (Amst) 7, 20–30. 41. Mazloum, N., Stegman, M.A., Croteau, D.L., Van Houten, B., Kwon, N.S., Ling, Y., Dickinson, C., Venugopal, A., Towheed, M.A., and Nathan, C. (2011) Identification of a chemical that inhibits the mycobacterial UvrABC complex in nucleotide excision repair. Biochemistry 50, 1329-1335. 42. Garbom, S., Forsberg, Å., Wolf-Watz, H., and Kihlberg, B.-M. (2004) Identification of novel virulence-associated genes via genome analysis of hypothetical genes. Infect. Immun. 72, 1333–1340.


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43. Graham, J.E., and Clark-Curtiss, J.E. (1999) Identification of Mycobacterium tuberculosis RNAs synthesized in response to phagocytosis by human macrophages by selective capture of transcribed sequences (SCOTS). Proc. Natl. Acad. Sci. U. S. A. 96, 11554–11559. 44. Darwin, K.H., and Nathan, C.F. (2005) Role for nucleotide excision repair in virulence of Mycobacterium tuberculosis. Infect. Immun. 73, 4581–4587. 45. Eldholm, V., Norheim, G., von der Lippe, B., Kinander, W., Dahle, U.R., Caugant, D.A., Mannsåker, T., Mengshoel, A.T., Dyrhol-Riise, A.M., and Balloux, F. (2014) Evolution of extensively drug-resistant Mycobacterium tuberculosis from a susceptible ancestor in a single patient. Genome Biol. 15, 490. 46. Truglio, J.J., Karakas, E., Rhau, B., Wang, H., DellaVecchia, M.J., Van Houten, B., and Kisker, C. (2006) Structural basis for DNA recognition and processing by UvrB. Nat. Struct. Mol. Biol. 13, 360-364. 47. Sambrook, J. and Russell, D.W. (2001) Molecular Cloning: A Laboratory Manual, 3rd Edition. Cold Spring Harbor, New York: Cold Spring Harbor Laboratory Press. 48. Dewhare, S.S., Umesh, T.G., and Muniyappa, K. (2015) Molecular and functional characterization of RecD, a novel member of the SF1 family of helicases, from Mycobacterium tuberculosis. J. Biol. Chem. 290, 11948-11968. 49. Hildebrand, E.L., and Grossman, L. (1999) Oligomerisation of the UvrB nucleotide excision repair protein of Escherichia coli. J. Biol. Chem. 274, 27885–27890. 50. Theis, K., Chen, P. J., Skorvaga, M., Van Houten, B., and Kisker, C. (1999) Crystal structure of UvrB, a DNA helicase adapted for nucleotide excision repair. EMBO J. 18, 6899-6907.


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51. Machius, M., Henry, L., Palnitkar, M., and Deisenhofer, J. (1999) Crystal structure of the DNA nucleotide excision repair enzyme UvrB from Thermus thermophilus. Proc. Natl. Acad. Sci. U.S.A. 96, 11717–11722. 52. Nakagawa, N., Sugahara, M., Masui, R., Kato, R., Fukuyama, K., and Kuramitsu, S. (1999) Crystal structure of Thermus thermophilus HB8 UvrB protein, a key enzyme of nucleotide excision repair. J. Biochem. 126, 986–990. 53. Webster, M.P., Jukes, R., Zamfir, V.S., Kay, C.W., Bagnéris, C., and Barrett, T. (2012) Crystal structure of the UvrB dimer: insights into the nature and functioning of the UvrAB damage engagement and UvrB-DNA complexes. Nucleic Acids Res. 40, 87438758. 54. Sohi, M., Alexandrovich, A., Moolenaar, G., Visse, R., Goosen, N., Vernede, X., Fontecilla-Camps, J.C., Champness, J., and Sanderson, M.R. (2000) Crystal structure of Escherichia coli UvrB C-terminal domain, and a model for UvrB-UvrC interaction. FEBS Lett. 465, 161-164. 55. Verhoeven, E.E., Wyman, C., Moolenaar, G.F., Hoeijmakers, J.H., and Goosen N. (2001) Architecture of nucleotide excision repair complexes: DNA is wrapped by UvrB before and after damage recognition. EMBO J. 20, 601-611. 56. Hsu, D.S., Kim, S.T., Sun, Q., and Sancar, A. (1995) Structure and function of the UvrB protein. J. Biol. Chem. 270, 8319-8327. 57. Yamagata, A., Masui, R., Kato, R., Nakagawa, N., Ozaki, H., Sawai, H., Kuramitsu, S., and Fukuyama, K. (2000) Interaction of UvrA and UvrB proteins with a fluorescent single-stranded DNA. Implication for slow conformational change upon interaction of UvrB with DNA. J. Biol. Chem. 275, 13235-13242.


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68. Thakur, R.S., Basavaraju, S., Khanduja, J.S., Muniyappa, K., and Nagaraju, G. (2015) Mycobacterium tuberculosis RecG protein but not RuvAB or RecA protein is efficient at remodeling the stalled replication forks: implications for multiple mechanisms of replication restart in mycobacteria. J. Biol. Chem. 290, 24119-24139. 69. Malta, E., Moolenaar, G.F., and Goosen, N. (2006) Base flipping in nucleotide excision repair. J. Biol. Chem. 281, 2184-2194. 70. Hall, M. C., and Matson, S. W. (1999) Helicase motifs: the engine that powers DNA unwinding. Mol. Microbiol. 34, 867-877. 71. Morimyo M., and Shimazu Y. (1976) Evidence that the gene uvrB is indispensable for a polymerase I deficient strain of Escherichia coli K-12. Mol. Gen. Genet. 147, 243–250. 72. Moolenaar, G.F., Moorman, C., and Goosen, N. (2000) Role of the Escherichia coli nucleotide excision repair proteins in DNA replication. J. Bacteriol. 182, 5706-5714. 73. Shizuya, H., and Dykhuizen, D. (1972) Conditional lethality of deletions which include uvrB in strains of Escherichia coli lacking deoxyribonucleic acid polymerase I. J. Bacteriol. 112, 676–681. 74. Siegel E.C. (1973) Ultraviolet-sensitive mutator mutU4 of Escherichia coli inviable with polA. J. Bacteriol. 113, 161–166. 75. DellaVecchia, M.J., Merritt, W.K., Peng, Y., Kirby, T.W., DeRose, E.F., Mueller, G.A., Van Houten, B., and London, R.E. (2007) NMR analysis of [methyl-13C]methionine UvrB from Bacillus caldotenax reveals UvrB-domain 4 heterodimer formation in solution. J. Mol. Biol. 373, 282-295.


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76. Waters, T.R., Eryilmaz, J., Geddes, S., and Barrett, T. E. (2006) Damage detection by the UvrABC pathway: crystal structure of UvrB bound to fluorescein-adducted DNA. FEBS Lett. 580, 6423–6427. 77. Patel, S.S., and Picha, K.M. (2000) Structure and function of hexameric helicases. Annu. Rev. Biochem. 69, 651–697. 78. Wong, I., and Lohman, T.M. (1992) Allosteric effects of nucleotide cofactors on Escherichia coli Rep helicase–DNA binding. Science 256, 350–355. 79. Chisty, L.T., Toseland, C.P., Fili, N., Mashanov, G.I, Dillingham, M.S., Molloy, J.E., and Webb, M. R. (2013) Monomeric PcrA helicase processively unwinds plasmid lengths of DNA in the presence of the initiator protein RepD. Nucleic Acids Res. 41, 5010-5023. 80. Yeung, A.T., Mattes, W.B., and Grossman, L. (1986) Protein complexes formed during the incision reaction catalyzed by the Escherichia coli UvrABC endonuclease. Nucleic Acids Res. 14, 2567-2582.

Legends to figures Figure 1. Expression and purification of MtUvrB, MtUvrBC146A and EcUvrB. Panels A-B, SDS-PAGE analysis showing induced expression of MtUvrB and MtUvrBC146A, and during different stages of their purification. Lane 1, standard protein markers; 2, uninduced wholecell lysate; 3, induced whole-cell lysate; 4, eluate from the Ni2+-NTA column; 5, eluate from Q Sepharose column; 6, Western blot of purified MtUvrB using anti-His antibodies. Panel C, SDS-PAGE analysis showing induced expression of EcUvrB and during different stages of its purification. Lane 1, standard protein markers; 2, uninduced whole-cell lysate;


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3, induced whole-cell lysate; 4, eluate from Q Sepharose column;5, eluate from Fast Performance Liquid Chromatography using Superdex-200; 6, eluate from Blue-Sepharose column; 7, Western blot of purified EcUvrB using anti-MtUvrB antibodies. Panels A-C: approximately 30 µg (lanes 2-3), 10 µg (lane 4), 8 µg (lane 5) and 6 µg (lane 5) of the indicated sample was resolved on SDS-PAGE and visualized by staining with Coomassie brilliant blue. Panel C, lanes 2-3, 30 µg; 4, 15 µg; 5, 8 µg and 6, 6 µg. The sizes of standard protein markers are indicated on the left in kDa. Western blots were performed with 5 µg of the indicated protein.

Figure 2. Determination of the molecular mass of MtUvrB. A, Chromatogram depicting the elution profile of markers (green line) and MtUvrB (red line). Graphical representation shows molecular weight as a function of Kav vs log molecular weight of marker proteins. Molecular weight of MtUvrB (~77 kDa) calculated by interpolation (depicted by red line in the inset). B, Chemical crosslinking of MtUvrB. Reactions were performed as described under “Experimental procedures.” Lane 1, standard markers; 2, MtUvrB incubated in the absence of crosslinker, 3-8); increasing concentration (0.005%, 0.0075%, 0.010%, 0.015%, 0.02%, and 0.03%) of glutaraldehyde. F, SEC-MALS data showing the elution volume vs molecular mass and UV absorbance at 280 nM. Data analysis was carried out by using ASTRA (Wyatt Technology Corporation) and molecular weight was deduced.

Figure 3. Binding of MtUvrB to various DNA substrates. Reaction mixtures contained 0.5 nM of the indicated 32P-labelled substrate in the absence (lane1) or presence of 0.05, 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1 and 1.2 µM of MtUvrB (lanes 2-13), respectively. The filled triangle on the top of each gel represents increasing concentration of MtUvrB. The


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Biochemistry 47

bracket and arrowhead on the right-hand side of each gel image denote DNA-MtUvrB complexes and free DNA, respectively. (A) fluorescein-ssDNA; (B) unmodified ssDNA; (C) fluorescein-dsDNA; (D) unmodified dsDNA; (E) splayed duplex DNA; (F) duplex with 3' overhang; (G) duplex with 5' overhang; (H) 3' flap; (I) 5' flap; (J) replication fork; (K) bubble containing dsDNA; (L) Holliday junction. (M), graphical representation of the extent of complex formation (in panels A-L) as a function of MtUvrB concentration. Each point represents the mean of the three independent experiments. The best-fit curve was obtained by subjecting the data sets to the non-linear regression analysis, in GraphPad PRISM (ver5.00), using the equation for one site specific binding with Hill slope.

Figure 4. MtUvrB exhibits intrinsic ATPase activity which is stimulated by DNA. Reaction mixtures contained either 200 nM DNA (panels (A) and (B)) or no DNA (panel (a), 200 pM [γ-32P]ATP, 30 µM cold ATP in the absence (lane 1) or presence of 20, 40, 80, 100, 120, 140 and 160 nM of MtUvrB (lanes 2–9), respectively. Panels: (A), in the absence of DNA; (B), in the presence of fluorescein-ssDNA; (C), in the presence of fluorescein-dsDNA. The yellow circle represents DNA containing fluorescein. The positions of [γ-32P]ATP and 32Pi is indicated on the right-hand side. (D) graphical representation of the extent of ATPase activity as a function of MtUvrB concentration. Each point is the mean of three independent experiments. Non-linear regression analysis (Michaelis-Menten) was applied to the data sets, using GraphPad PRISM ver5.00, for obtaining the best-fit curve.

Figure 5. Determination of the kinetic parameters of MtUvrB ATPase activity. Assay was performed with a constant amount of MtUvrB (60 nM) in the absence (panel A) or presence


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of 200 nM fluorescein-ssDNA (panel B), 10 mM MgCl2 and 2, 5, 10, 15, 20, 30 and 40 µM [γ-32P]ATP (lanes 2-16), respectively. A working ATP standard solution (500 µM cold ATP and 2.7 pM [γ-32P]ATP as a tracer) was prepared on the day of the assay. Lane 1 (in panel A and B) represents the reaction mixture containing 20 µM ATP in the absence of MtUvrB. Reaction mixtures were incubated for 60 min at 37 °C. Reaction products were separated as described under Experimental procedures. (B) and (C) show 1/[v] versus 1/[S] in the form of a Lineweaver-Burk plot in the absence and presence of fluorescein-ssDNA, respectively. For each point, initial velocities were determined from multiple time courses over time ranges giving linear hydrolysis of ATP. (E) percent hydrolyses were plotted as functions of ATP concentration and fitted to the Michaelis-Menten equation.

Figure 6. MtUvrB unwinds DNA replication/repair structures. Assay was performed with 0.5 nM of the indicated 32P-labelled DNA substrate in the absence (lane 1) or presence of 10, 20, 35, 50, 65, 80, 100 or 120 nM MtUvrB (lanes 3-10), respectively. Reaction mixtures were preincubated at 37 ̊C for 5 min. The reaction was started by the addition of 3 mM ATP and 5 nM of cold DNA as a trap for complimentary strand of radiolabelled strand. Reaction mixtures were further incubated at 37 ̊C for 90 min, and assayed as described under “Experimental procedures.” Panels (A-I), gel images depicting MtUvrB catalyzed unwinding of the indicated DNA substrate. Panel (J), Graphical plot showing the extent of unwinding of DNA substrates in panels A-I plotted versus increasing concentrations of MtUvrB. Vertical bars represent standard deviation of three independent experiments. Panels (A-I): Lane 1, substrate alone; 2 heat-denatured substrate. Filled triangles on top of the gel images represent increasing concentrations of MtUvrB. Asterisk (*) denotes the 32P-


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Biochemistry 49

labelled 5’- phosphate end. The structure and positions of the substrate DNA and unwound DNA product are indicated on the right hand side.

Figure 7. Effect of the length of ssDNA tails on MtUvrB helicase activity. Assay was performed with 0.5 nM of the indicated 32P-labelled DNA substrate in the absence (lane 1) or presence of 5, 10, 20, 30, 40, 50, 60, or 80 nM of MtUvrB (lanes 3-10), respectively. Lane 2 contained heat denatured substrate. Reaction mixtures were preincubated at 37 ̊C for 5 min. The reaction was started by the addition of 3mM ATP and 5nM of cold DNA as a trap for complimentary strand of radiolabelled strand. Reaction mixtures were further incubated at 37 ̊C for 90 min and assayed as described under “Experimental procedures.” Panels A-F, gel images depicting MtUvrB catalyzed unwinding of the indicated DNA substrate. Panel G, graphical representation of the extent of unwinding of DNA substrates in panels A-F plotted against increasing concentrations of MtUvrB. Vertical bars denote standard deviation from three independent experiments. Filled triangles on top of the gel images represent increasing concentrations of MtUvrB. Asterisk (*) denotes the 32P-labelled end. The structure, length of each arm and positions of the substrate DNA and unwound DNA product are indicated on the right hand side.

Figure 8. Kinetics of DNA unwinding catalyzed by MtUvrB. Reaction mixtures containing 0.5 nM of DNA substrate and 80 nM MtUvrB was incubated for 5, 10, 15, 20, 25, 30, 35, 40 ,45, 55, 65 and 75 min (lanes 3–14), respectively. Lane 1, substrate alone; 2, heatdenatured substrate. (A) unwinding of splayed duplex; (B) 3'-flap; and (C) 5'-flap. (D) graphical representation of the kinetics of unwinding. The intensity of bands in panels was


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quantified using UVI-BandMap software ver. 94.0 and plotted using Graphpad Prism ver. 5.0. The data points represent the mean of three independent experiments.

Figure 9. MtUvrB helicase activity is sensitive to the duplex length. Assay was performed with 0.5 nM of the indicated 32P-labelled DNA substrate in the absence (lane 1) or presence of 10, 20, 35, 50, 65, 80, 100 or 120 nM of MtUvrB (lanes 3-10), respectively. Lane 2 represents heat denatured substrate. Reaction mixtures were preincubated at 37 ̊C for 5 min. The reaction was started by the addition of 3 mM ATP and 5 nM of cold DNA as a trap for complimentary strand of radiolabelled strand. Reaction mixtures were further incubated at 37 ̊C for 90 min and assayed as described under Experimental procedures. Panels A-H, gel images depicting MtUvrB catalyzed unwinding of the indicated DNA substrate. Panel I, graphical plot shows the extent of unwinding of DNA substrates in panels A-H plotted versus increasing concentrations of MtUvrB. The intensity of bands was quantified and plotted against the indicated concentrations of MtUvrB. Vertical bars represent standard deviation of three independent experiments. Filled triangles on top of the gel images represent increasing concentrations of MtUvrB. Asterisk (*) denotes the 32P-labelled end. The structure, length of each arm and positions of the substrate DNA and unwound DNA product are indicated on the right hand side.

Figure 10. MtUvrB unwinds modified splayed duplexes. Reactions contained a 0.5 nM concentration of 32P-labelled splayed duplexes with either 3′-3′ linkage (A), ′ 5′-5′ linkage (B) or 5'-5'/3'-3'. Reactions were performed in the absence of MtUvrB (lanes 1) or in the presence of 10, 20, 30, 40, 50, 60, 70, and 80 nM MtUvrB (lanes 3-10), respectively, in the


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Biochemistry 51

assay buffer containing 3 mM ATP and 1 mM MgCl2. Lane 2, heat-denatured substrate. The filled triangle at the top of the gel image denotes increasing concentrations of MtUvrB. (D) Quantitative data for the ability of MtUvrB to unwind the modified splayed duplexes. Data represent the mean of at least two independent experiments with S.D. values indicated by error bars.

Figure 11. MtUvrB possesses unipolar ssDNA translocase activity. Panels A-B: gel images depicting MtUvrB translocation over ssDNA in the presence of streptavidin. Assay was performed with 1 nM 32P-labelled DNA substrate in presence of 50, 100, 200, 350, 500, 650, 800 and 1000 nM MtUvrB (lanes 3-10), respectively. Lane 1, reaction mixture in the absence of protein. Reaction mixtures were pre-incubated at 37 ̊C for 5 min. The reaction was started by the addition of 3.5 mM ATP and assayed as described under Experimental procedures. Filled triangles on top of the gel images indicate increasing concentrations of MtUvrB. A yellow triangle on substrate DNA ends denotes the position of biotinylated nucleotide. Asterisk (*) denotes the 32P-labeled end. The positions of the displaced DNA and streptavidin bound to DNA are indicated on the right hand side. (C) graphical plot shows the extent of displacement of streptavidin versus increasing concentrations of MtUvrB. Figure 12. Panels A-C, gel images depicting EcUvrB and MtUvrB catalyzed unwinding of the indicated DNA substrate. Assay was performed with 0.5 nM of the indicated 32Plabelled DNA substrate in the absence (lane 1) or presence of 0.1, 0.2, 0.35, 0.5, 0.65, 0.8, 1 or 1.2 µM EcUvrB (lanes 2-10) and 80 nM of MtUvrB (lane 11), respectively. Reaction mixtures were preincubated at 37 ̊C for 5 min. The reaction was started by the addition of 3


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mM ATP and 5 nM of cold DNA as a trap for complimentary strand of radiolabelled strand. Reaction mixtures were further incubated at 37 ̊C for 90 min and assayed as described under Experimental procedures. Panels D-E, assay was performed with 0.5 nM of the indicated 32

P-labelled splayed-duplex DNA substrate in the absence (lane 1) or presence (lanes 3-10),

of 0.1, 0.2, 0.35, 0.5, 0.65, 0.8, 1 or 1.2 µM of MtUvrBC146A and MtUvrB in panel C and D, respectively. F, Graphical representation of the extent of unwinding of DNA substrates in panels A-D plotted versus increasing concentrations of protein. Vertical bars represent standard deviation of three independent experiments. Panels A-D: Lane 1, substrate alone; 2, heat-denatured substrate. Filled triangles on top of the gel images represent increasing concentrations of MtUvrB. An asterisk (*) denotes the 32P-labelled 5’- phosphate end. The structure and positions of the substrate DNA and unwound DNA product are indicated on the right hand side.


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Biochemistry 53

Table 1. Construction of DNA substrates (the asterisk denotes the 5' radiolabelled end)


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Table 2. Dissociation Constants for MtUvrB-DNA Complexes

Substrate

K d (nM)

Fluorescein-ssDNA

136.8 ± 4.46

Unmodified ssDNA

285.4 ± 3.19

Fluorescein-dsDNA

ND

Unmodified dsDNA

ND

Splayed duplex

389.5 ± 2.69

3' overhang

482 ± 2.51

5' overhang

530.5 ± 1.72

3' flap

365.4 ± 2.69

5' flap

1427.5 ± 2.17

Replication fork

1716.2 ± 3.30

Bubble-containing dsDNA

2247.9 ± 0.23

Holliday junction

ND

N.D.: not determined.


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Figure 1

Figure 2


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Figure 3


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Figure 4


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Figure 5


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Figure 6


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Figure 7


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Figure 8

Figure 9


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Figure 10

Figure 11


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Figure 12

A

B

EcUvrB

C ∆

EcUvrB

C ∆

* * *

* 1

2

3 4

5

C

6 7

8

9 10 11

1

3

4

5

D

EcUvrB

C ∆

2

C

6 7

8

9 10 11

MtUvrBC146A

∆

*

* * E

1

2

3 4

5

6 7

8

1

9 10 11

2

3

F

MtUvrB

C ∆

*

* * 1

2

3

4

5

6

7

8

9 10


4

5

6 7

8

9 10

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For Table of Contents Use Only

Mycobacterium tuberculosis UvrB is a robust DNA-stimulated ATPase that also possesses structure-specific ATP-dependent DNA helicase activity Manoj Thakur, Mohan B. J. Kumar and Kalappa Muniyappa* Department of Biochemistry, Indian Institute of Science, Bangalore 560012, India


Mycobacterium tuberculosis UvrB Is a Robust DNA-Stimulated

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