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Structures of the catalytic domain of bacterial primase DnaG in complexes with DNA provide insight into key priming events Caixia Hou, Tapan Biswas, and Oleg Tsodikov Biochemistry, Just Accepted Manuscript • DOI: 10.1021/acs.biochem.8b00036 • Publication Date (Web): 20 Mar 2018 Downloaded from http://pubs.acs.org on March 22, 2018

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Biochemistry

Structures of the catalytic domain of bacterial primase DnaG in complexes with DNA provide insight into key priming events Caixia Hou,a Tapan Biswas,b and Oleg V. Tsodikova,* a

Department of Pharmaceutical Sciences, College of Pharmacy, University of Kentucky, Lexington, KY,

40536, USA. b Department of Chemistry and Biochemistry, University of California, San Diego, La Jolla, CA, 92093, USA.

KEYWORDS: DNA replication, Replisome, Crystal structure, DNA binding, Tuberculosis

ACS Paragon Plus Environment

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ABSTRACT

Bacterial primase DnaG is an essential nucleic acid polymerase that generates primers for replication of chromosomal DNA. The mechanism of DnaG remains unclear, due to the paucity of structural information on DnaG in complexes with other replisome components. Here we report the first crystal structures of noncovalent DnaG-DNA complexes, obtained with the RNA polymerase domain of Mycobacterium tuberculosis DnaG and various DNA ligands. One structure, obtained with ds DNA, reveals interactions with DnaG as it slides on ds DNA and suggests how DnaG binds template for primer synthesis. In another structure, DNA in the active site of DnaG mimics the primer, providing insight into mechanisms for the nucleotide transfer and DNA translocation. In conjunction with the recent cryo-EM structure of the bacteriophage T7 replisome, this study yields a model for primer elongation and hand-off to DNA polymerase.


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Biochemistry

INTRODUCTION

Primases are DNA-dependent RNA polymerases that generate primers for chromosomal DNA replication by using single-stranded (ss) DNA as a template. In contrast, replicative polymerases cannot initiate DNA replication by using ss DNA as a template; therefore, primase activity is required. In bacteria primer synthesis is carried out by the DNA-dependent RNA polymerase DnaG.1 DnaG is a modular protein composed of three independently folded domains: the N-terminal zinc binding domain (ZBD), which was implicated in DNA binding, initiation of priming and the length control of Okazaki fragments,2,

3

the large central RNA

polymerase domain (RPD) containing the catalytic site for the nucleotide transfer (Fig. S1),4-6 and the C-terminal replicative helicase binding domain (HBD). The RPD, which is conserved in bacteria and bacteriophages, consists of three subdomains organized in a linear fashion: an α/βfold containing N-terminal subdomain (poorly conserved in phages), the central catalytic subdomain containing a TOPRIM fold present in type IA and type II topoisomerases, nucleases and other proteins and the C-terminal helical subdomain.4, 5 The essential function of DnaG and its unique structure make it an attractive, yet underexploited, target for antibacterial drug discovery.7-17 Our recent studies functionally characterized DnaG from a M. tuberculosis (Mt), developed a coupled colorimetric primase-phosphatase activity assay, and utilized it in a pilot high-throughput screening, which led to discovery of low-µM inhibitors of DnaG.10 The paucity of structural information on complexes of DnaG with its interacting protein and nucleic acid replisome components leave many mechanistic questions about the DnaG function unanswered. The structure of the complex of the replicative helicase from B. stearothermophilus with the cognate HBD of DnaG has revealed how DnaG docks onto the helicase.18 The locations


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of the HBDs on the helicase are consistent with the structures of the primase-helicase of phage T7 (gene product gp4; T7gp4), in which the primase (homologous to the ZBD-RPD of DnaG) and the helicase are a part of the same polypeptide chain.19, 20 Crystal structures of complexes of the RPD of S. aureus DnaG with nucleotides led the the proposal that the nucleotides mimicked an incoming NTP during synthesis.6 Crystallization of DnaG-DNA complexes has been extremely challenging. The only example, the structure of the RPD of E. coli DnaG chemically crosslinked to a single-stranded oligonucleotide suggested how the RPD may interact with a region of the template.21 Finally, a recent cryo-EM structure of a T7 replisome demonstrated how two DNA polymerases were positioned on the helicase-primase bound to a DNA substrate mimicking the replication fork.22 Unfortunately, only the protein components, but not the DNA, were observed in this complex. In pursuit of improving our understanding of function of DnaG, we obtained crystal structures of MtDnaG alone and in complexes with DNA. These are the first crystal structures of non-covalent DnaG-DNA complexes. These structures yield important insight into the mechanism of nucleotide transfer by the primase, and allow us to construct a nucleoprotein model of the lagging strand replisome, clarifying a mechanism for primer handover to the DNA polymerase.

MATERIALS AND METHODS

Cloning and purification of the RPD, the ZBD, the full-length MtDnaG and its mutants. The DNA region encoding the RPD domain of MtDnaG from Mt H37Rv (residues 112-432) was amplified by PCR by using the plasmid encoding full-length MtDnaG10 as a template with primers

5’-ATCAACCACATATGGGCAGTCGCAGCAGGCTGC-3’


and

5’-

4

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Biochemistry

ATCCTCGAGTCATGCGACGTCGGCCCACCCG-3’, and cloned between NdeI and XhoI sites of a the same modified pET-19b vector.23 The coding regions for MtDnaG ZBD (residues 1-109) was amplified with primers 5’-AGTTAGCACATATGTCCGGCCGGATCTCCG-3’ and 5’CCGCTCGAGTCAGCGCTGCACGCTG-3’, and that for the MtDnaG ZBD-RPD didomain construct (residues 1-432) was amplified with the forward primer for the ZBD construct and the reverse primer for the RPD construct; the ZBD and the ZBD-RPD constructs were cloned analogously to the RPD. The mutants R190A, R199A and Y233F were generated by using a QuikChange kit (Agilent Genomic) from the full-length MtDnaG construct with the primers listed in Table S3. In the expressed protein products, the N-terminal decahistidine tag is cleavable by PreScission protease (GE Healthcare). The expression and purification of the RPD, the ZBD, the ZBD-RPD, full-length MtDnaG and its mutants were carried out as previously described for the full-length MtDnaG.10 In brief, Escherichia coli BL21(DE3) cells transformed with the expression vector were induced with 0.5 mM IPTG at the attenuance of 0.4 at 600 nm and incubated with shaking for 16 hours at 18 °C. The cell pellet was resuspended in the lysis buffer (40 mM Tris-HCl pH 8.0, 400 mM NaCl, 10% v/v glycerol, 2 mM MgCl2 and 2 mM βmercaptoethanol) and lysed by sonication. The protein was then purified by Ni2+-chelating chromatography on a HiTrap-IMAC HP column (GE Healthcare), following the manufacturer’s instructions. The tag was cleaved with PreScission protease overnight at 4 °C, and the digested protein was purified on a size-exclusion S-200 column (GE Healthcare) equilibrated with 40 mM Tris-HCl pH 8.0, 200 mM NaCl and 2 mM β-mercaptoethanol. The fractions containing MtDnaG were concentrated to 10 mg/ml for crystallization and DNA binding assays. Protein crystallization, data collection, structure determination and refinement. The crystallization experiments were carried out by vapor diffusion in hanging drops at 21 °C. For


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crystallization of the RPD alone, the drops were set up by mixing 1 µL of RPD and 1 µL of the reservoir solution (50 mM Tris pH 8.5, 1.85 M ammonium sulfate, and 5% v/v PEG 400 and 2 mM MnCl2). The crystals were gradually transferred to the cryoprotecting buffer (50 mM Tris pH 8.5, 1.85 M ammonium sulfate and 26.5% sucrose) and frozen by quick immersion into liquid nitrogen. The ds DNA oligomer for crystallization and DNA binding experiments were prepared by heating a mixture of 1 mM of a single-stranded 12-mer 5’-GACCGGAAGTGG-3’ (IDT) and 1 mM of its complement in 10 mM Tris pH 8.0 and 50 mM NaCl to 95 °C and then cooling it to 4 °C over 30 min. The overhang DNA ligands were prepared analogously, by annealing 13-mer 5’TGACCGGAAGTGG-3’ with 12-mer 5’-CCACTTCCGGTC-3’ for the 1-nt overhang DNA and 14-mer 5’-TATCGTCCCGCCTC-3’ with an 8-mer 5’-GAGGCGGG-3’ for the 6-nt overhang DNA. For co-crystallization with these DNA ligands, the RPD was concentrated to 25 mg/mL and mixed with 1 mM of each DNA ligand at a 1:1.5 protein:DNA ratio. The hanging drops for the RPD-ds DNA complexes were set as described above for the RPD alone, with the reservoir solution composed of 100 mM Hepes pH 7.0, 7% w/v PEG 4000. The crystals were gradually transferred to the cryoprotecting solution (100 mM Hepes, pH 7.0, 7% PEG 4000, 32% sucrose) and frozen by quick immersion into liquid nitrogen. For the MtDnaG RPD-1-nt overhang DNA complexes, the reservoir solution was 100 mM Hepes pH 7.0, 6% PEG 4000, 5 mM SrCl2. For the RPD- 6-nt 5’-overhang DNA complex, the reservoir solution was 100 mM sodium citrate pH 6.0, 5% w/v PEG 4000 and 5 mM SrCl2. The crystals of MtDnaG RPD-overhang DNA complexes were cryoprotected by gradually transferring them to the solutions with the same compositions as the respective reservoir solutions, each additionally containing 30% glycerol prior to freezing.


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Biochemistry

The X-ray diffraction data were collected at 100 K at beamline 22-ID of the Advanced Photon Source at the Argonne National Laboratory (Argonne, IL). The data were processed with HKL-2000

24

. The initial model of the RPD alone was obtained by sequential molecular

replacement with the N-terminal subdomain of the E. coli DnaG RPD (PDB code 1DDE, residues 115-249; 4) followed by the central subdomain of this protein (residues 260-363) as search models, by using Phaser.25 The missing parts of the structure were then built into the unambiguous Fo-Fc electron density in the course of overall iterative model building and refinement with Coot26 and Refmac27, respectively. The crystal structure of the MtDnaG RPD- ds DNA complex was determined by molecular replacement with the structure of the MtDnaG RPD as a search model. The structures of MtDnaG RPD - overhang DNA complexes were determined by molecular replacement with the MtDnaG RPD part of the structure of MtDnaG RPD - ds DNA as a search model. The DNA in all cases was built into the Fo-Fc electron density, and further building and refinement proceeded as described above for the structure of the RPD alone. The data collection and refinement statistics are given in Tables S1 and S2. DNA binding assays. DNA binding was measured by monitoring fluorescence anisotropy of a 6-carboxyfluorescein (6-FAM) probe on (6-FAM)-5’-labeled single-stranded (ss) 12-mer 5’CCACTTCCGGTC-3’ or double-stranded (ds) DNA, where this ss oligomer was annealed to its complement. The single-stranded oligomers were purchased from IDT. The DNA was annealed at 1.1:1 ratio of unlabeled:labeled oligomer, as described previously.28 Full-length MtDnaG or its RPD at concentrations specified in Results were titrated into DNA (at the constant concentration of 50 nM). The binding buffer was 20 mM CAPS, pH 8.8; 2 mM MnCl2 and 50 mM NaCl. The binding mixture was incubated for 10 min at 22 °C. Fluorescence was measured on a SpectraMax M5 microplate reader (Molecular Devices), at excitation and emission wavelengths


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of 494 nm and 525 nm, respectively. The best-fit values of the equilibrium binding constants (Kd) were obtained by nonlinear regression fitting of the 1:1 isotherm as described previously.29 Primase activity assays. The primase nucleotidyl transfer activity was measured by the coupled colorimetric primase-inorganic pyrophosphatase assay in the presence of Mn2+ as reported10, except that 1 µM full-length MtDnaG (wild-type or a mutant, as specified), 1 µM ZBD-RPD, RPD, or 1 µM RPD and 1 µM ZBD were used in the assay. The signal measured in the absence of MtDnaG was subtracted, and then the activity was normalized to that of the wildtype full-length MtDnaG.

RESULTS

DNA binding and primase activities of full-length MtDnaG and its RPD. To bind the replication fork and catalyze primer synthesis, DnaG must engage with single-stranded DNA, a DNA-RNA hybrid, and likely with double-stranded DNA, when not bound at the replication fork. Therefore, the interacting surfaces of DnaG must be distinct or versatile, in order to bind various forms of nucleic acid structure. We overexpressed in E. coli and purified recombinant full-length MtDnaG and its RPD and measured their binding affinity to ss and ds 12-mer DNA oligomers. We monitored the changes in anisotropy of fluorescence of a 6-carboxyfluorescein label on the 5’-end of one of the DNA strands, upon titrating full-length MtDnaG or its RPD into the ss or the ds DNA (Fig. 1, panels A and B). Equilibrium constants (Kd) for binding of MtDnaG and its RPD to the ss DNA are 0.18 ± 0.03 µM and 0.83 ± 0.16 µM, respectively, indicating that the RPD bears important determinants of the affinity of MtDnaG to ss DNA. Binding of the full-length MtDnaG (Fig. 1A) to the ds DNA was ~2-fold weaker (Kd = 0.33 ± 0.05 µM) than that to the ss DNA, and binding of the RPD to the ds DNA (Fig. 1B)


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Figure 1

A 0.35

Fluorescence anisotropy

0.30 0.25 0.20 0.15 0.10

ss DNA, Kd = 0.18 ± 0.03 µM ds DNA, K d = 0.33 ± 0.05 µM

0.05 0

500

1000 1500 [full-length MtDnaG], nM

2000

2500

B 0.35


0.30 0.25 0.20 0.15 0.10

ss DNA, Kd = 0.83 ± 0.16 µM ds DNA, K d = 0.50 ± 0.08 µM

0.05 0

500

1000 1500 [MtDnaG RPD], nM

C

2000

2500

100

80

Relative activity, %

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

Biochemistry

60

40

20

0 MtDnaG

f.l. wt

f.l. wt

RPD

ZBD RPD+ZBD ZBD-RPD

DNA

Figure 1. DNA binding and primase activities of MtDnaG and its domains. Equilibrium binding titrations of (A)


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full-length MtDnaG and (B) its RPD to ss (open circles) and ds (filled squares) 12-mer DNA oligomers. The curves are the best-fit 1:1 binding isotherms generated for the Kd values indicated in the insets. C. Primase activity of fulllength (f. l.) MtDnaG (black bars), its Y233F mutant (a white bar), the RPD and the ZBD (light gray bars), a 1:1 RPD:ZBD mixture (a dark gray bar) and the ZBD-RPD didomain, measured by the primase-inorganic pyrophosphatase coupled assay. All the data are the averages of independent triplicate experiments.

(Kd = 0.50 ± 0.08 µM) was comparable to its binding to the ss DNA. These observations indicate that the RPD is a dominant contributor of the affinity of MtDnaG to ds DNA. Since the MtDnaG RPD was the major contributor to DNA binding, we asked if the RPD was sufficient for the nucleotidyl transfer activity of MtDnaG. The primase activity was measured by our recently developed coupled colorimetric assay detecting inorganic pyrophosphate liberated upon nucleotide transfer.10

The RPD yielded a signal that was similar, within the experimental

uncertainty, to that from full-length MtDnaG in the absence of DNA (Fig. 1C), indicating that the RNA synthesis by the RPD was significantly or completely impaired. This result indicated that the missing domains, ZBD and/or HBD, were indispensable for the catalytic activity of MtDnaG. Indeed, the ZBD-RPD construct lacking only the C-terminal HBD displayed ~50% of the activity of the full-length enzyme, showing that the ZBD is necessary for the enzymatic activity, although it is not sufficient for restoring the full activity (Fig. 1C). These data also show that the lack of the HBD impairs the primase activity. We then tested whether the activity of the RPD could be rescued (to the level of the RPD-ZBD construct) by adding the purified ZBD (residues 1-109). While the ZBD alone was itself catalytically inert, we observed a marked increase in activity upon addition of the equimolar amount of purified ZBD (Fig. 1C). Crystal structure of the RPD of MtDnaG. To elucidate the structural basis for the ss and ds DNA binding by the RPD of MtDnaG, we determined the crystal structure of the MtDnaG RPD at the resolution of 2.85 Å (Fig. 2A). Sequential molecular replacement with the central


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Biochemistry

(TOPRIM) and the N-terminal subdomains of the RPD of E. coli DnaG (PDB ID: 1DDE;4) was required to obtain a partial structure of the RPD of MtDnaG, due to subtle differences in subdomain orientations in this RPD. The helical C-terminal subdomain was then built into the difference electron density map. This subdomain is less conserved in bacteria than the other

A

N

C

B

C Y233 β2

R199 K232 R221

R190 β3

Y143 M229 R147

Figure 2. The crystal structure of the RPD of MtDnaG. The N-terminal, the TOPRIM, and the C-terminal subdomains are colored pale turquoise, pale yellow and dark blue, respectively. The bound sulfate ions are shown as sticks. A. An overall view of the structure. The omit Fo-Fc map (green mesh) contoured at 3σ indicates bound sulfate ions. B. A zoomed-in view of the sulfate ion bound in the active site. C. A zoomed-in view of the sulfate bound to the N-terminal subdomain. In panels B and C, the residues interacting with the sulfate ions are shown as dark teal sticks.


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subdomains, and it is absent in bacteriophage T7 (Figs. S1 and S2). The lengths and orientations of numerous secondary structural elements in the N-terminal and TOPRIM subdomains, especially loop regions, are unique to MtDnaG. Two sulfate ions from the crystallization solvent were bound to the MtDnaG RPD (Fig. 2). One sulfate ion was bound in the vicinity of the active site, stabilized by salt bridges with conserved basic residues in the interface between the N-terminal and the TOPRIM subdomains. This ion is stabilized by salt bridges with N-terminal subdomain residues Arg147, Arg221 and Lys232 and hydrogen bonds with Tyr143 and Tyr233. All of these residues are conserved in bacteria (Fig. S1). The basic residues are also present in T7, but helix α2 containing Tyr143 is absent in T7, and in place of Tyr233 T7 contains a Phe.19 This sulfate ion binding site matches closely the location of the γ-phosphate group of the ATP in the crystal structure of S. aureus DnaG RPD-ATP complex6 (PDB ID: 4EDG; Fig. S2C). The second sulfate ion was bound in the basic groove on the surface of the N-terminal subdomain (Fig. 2C). This ion is held by salt bridges with Arg190 (in β strand β2) and Arg199 (in the loop connecting β3 and β4) and a steric interaction with Met229. The protein regions containing these residues are less conserved in sequence (Fig. S1) and structure (Fig. S2) than that involved in binding the other sulfate ion. Despite this variability, Arg199 is highly conserved, and an Arg can be found in the same or next position as Arg190 in the sequence alignment in some bacteria. T7 primase lacks these secondary structural elements altogether, but aromatic (Tyr66 and Tyr106) and basic (Lys131) residues from the differently placed N-terminal loop and the β-sheet of the N-terminal domain of the T7 primase RPD (Fig. S2D) appear to form an analogous surface. The location and the conservation of the sulfate binding sites strongly suggest that the two sulfate ions mimic phosphate groups of an incoming NTP or nucleic acid bound to MtDnaG.


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Biochemistry

Crystal structures of the RPD of MtDnaG in complex with double-stranded DNA. Crystal structures of noncovalent DnaG-DNA complexes have been elusive. Similar affinities of the RPD of MtDnaG to ds and ss DNA strongly suggested that ds DNA could be used to cocrystallize MtDnaG with DNA. Indeed, we obtained two crystal structures (at resolutions of 2.95 Å and 3.3 Å; Tables S1 and S2): one is a complex of the RPD with a ds 12-mer of the same sequence as that used in the DNA binding assays and the other with a ds 12-mer of the same sequence, but with one of the two strands longer by 1-nt at the 5’-end. These two crystal forms were similar to each other and entirely different from that of the crystals of the RPD alone. In both crystal structures, two molecules of the RPD and one DNA molecule were present in the A

B

3’

5’

R193 2

C 5

N

3

1

4

E230

R190 H194

M229 M197 W166

Figure 3. The crystal structure of the MtDnaG RPD bound to the ds 12-mer DNA. The DNA is shown in orange. A. An overall view of the structure. B. A zoomed-in view of the RPD-DNA interface, with the DNA interacting residues shown as dark teal sticks.

asymmetric unit. The conformations of the RPDs in these crystals are all very similar to each other and to that of the RPD crystallized without DNA, except for minor changes in the orientation of the C-terminal subdomain and the DNA interacting regions. The DNA was bound similarly to the N-terminal subdomain of one of the two RPDs in both structures (Fig. 3A, Fig. S3) and was involved in crystal packing interactions with another RPD molecule in the crystals


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(Fig. S4). In both structures, the RPD interacted only with one of the two DNA strands, in a similar fashion. The absence of protein interactions with one of the two strands explains the similar binding affinities of the RPD to the ds and ss DNA 12-mers. The majority of the proteinDNA interactions are with the DNA backbone, which is well defined by the difference density in both structures (e.g., Fig. S3). Possibly as a consequence of the lack of sequence specific interactions and due to distortions of the strand bound to the protein, the other strand was partially disordered in the structure with ds DNA and completely disordered in the structure with the DNA containing a 1-nt overhang. These DNA binding features strongly suggest that the region of the DNA strand interacting with the protein resembles a region of the template strand during replication. The DNA backbone of this interacting strand forms a network of salt bridges, hydrogen bonds and hydrophobic interactions with the RPD (Fig. 3B). The 5’-phosphate group of this region forms a salt bridge with His194. Phosphate group 2 is sandwiched by salt bridges with His 194 and Arg193, while the ribose ring of this nucleotide is in a nonpolar contact with Trp166. Phosphate 3 forms a hydrogen bond with the backbone amide of Arg193, and the C5’ backbone methylene group of this nucleotide contacts Met197. Phosphate 4 forms a salt bridge with Arg190, and the respective C5’ methylene is engaged in a hydrophobic interaction with Met229. Finally, phosphate 4 forms a hydrogen bond with a backbone amide of Glu230, whereas the aliphatic portion of this side chain contacts the respective C5’ methylene. This phosphate is positioned similarly to the second sulfate ion in the structure of the free RPD (Fig. 2B), confirming that this sulfate is a marker of DNA binding. A minor positional difference between this sulfate and the DNA phosphate must be a consequence of the restraints of the geometry of the ds DNA, whereas the the unrestrained sulfate ion likely indicates the path of the bound ss DNA more closely. In a previously reported crystal structure of a ss DNA oligomer crosslinked


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Biochemistry

to a Cys introduced into the RPD of E. coli DnaG in the vicinity of the active site,21 the ss DNA is bound in a similar location and with the same polarity.21 Our structure is free from a potential DNA polarity bias that could have resulted from the geometric constraints of the crosslink. The lack of the sequence conservation of the interacting residues (Fig. S1) and the resulting structural differences between the DNA interacting regions of the RPDs between Mt and E. coli necessitated different protein-DNA contacts (Fig. S1). Nevertheless, structural analogs of Trp166 and Arg190 (Trp165 and Val189 in E. coli) are also engaged in interactions with DNA in E. coli. Mutating a residue that interacts with DNA in both structures, Trp165 (Trp166 in Mt) to an Ala in E. coli DnaG abolished its binding to ss DNA and its primer synthesis activity,21 strongly suggesting that this protein-DNA interface is functionally important in MtDnaG as well. In order to probe the observed protein-DNA interface in MtDnaG directly, we measured the effect of two mutations, Arg190Ala and Arg199Ala, on binding to ss and ds 12-mer DNA and on the RNA synthesis activity of MtDnaG. Arg190 is not conserved outside of mycobacteria (Fig. S1), even though in E. coli DnaG, Val189 at this position interacts with DNA. A nearby Arg199 is conserved (with the exception of A. aeolicus) and is predicted to interact with DNA based on its interaction with the bound sulfate ion. Mutation of this residue in E. coli DnaG (Arg199) to an Ala led to a strong reduction in the RNA synthesis activity of the primase, although, oddly, the DNA binding affinity of the mutant was ~3-fold higher.21 We observed that both Arg190Ala and Arg199Ala mutants bound both short ss and ds more weakly than the wild-type MtDnag did (Fig. 4, panels A and B). For both ds and ss DNA, the Arg190Ala mutation (the residue observed to interact with ds DNA in our crystal structure) in both cases had a more deleterious effect on binding (a ~3-fold decrease in binding affinity). Similarly, the two mutants displayed comparably impared enzymatic activity (Fig. 4C), although this effect was not as pronounced,


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likely due to the requirement for higher DNA concentration in this assay (1 µM) than that in the DNA binding assays (50 nM). These experiments validate the DNA binding surface observed in


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Figure 4

A 0.28 wild-type

ds DNA Fluorescence anisotropy

0.26 0.24 R199A 0.22 0.20 R190A

0.18 0.16 0.14 0.12 0.10 0.0

0.5

1.0 1.5 [MtDnaG], µM

2.0

B 0.30

0.25 Fluorescence anisotropy

2.5

wild-type

ss DNA

R199A

0.20 R190A 0.15

0.10

0.05 0.0

0.5

C


2.0

2.5

100

80


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Biochemistry

60

40

20

0 MtDnaG

wild-type

R199A

R190A

Y233F

Figure 4. DNA binding and primase activities of MtDnaG mutants R190A and R199A. Equilibrium binding titrations of wild-type full-length MtDnaG (open circles) and its mutants R190A (open squares) and R199A (open circles) to (A) ds and (B) ss 12-mer DNA oligomers. The data represent two independent experiments. C. Primase


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Page 18 of 38

activity of wild-type full-length MtDnaG and its mutants: R190A, R199A and Y233F. The data are averages of independent triplicate measurements.

the crystal structures and provide direct evidence that this surface is used for binding both ds and ss DNA in a functionally significant manner. Crystal structure of the RPD of MtDnaG bound to DNA mimicking a primer in the active site. Mechanistic details of nucleotide transfer and DNA binding in the active site of DnaG have remained elusive, due to insufficient structural information. Our crystal structure of the RPD-ds DNA complex unambiguously reveals a key ds/ss DNA binding site on the Nterminal subdomain of the RPD. Its dominant contribution to binding ss and ds DNA in the context of the full-length MtDnaG suggest that this site is used by DnaG to bind the template and slide along ds DNA when idle, to locate the replisome. This binding, to only one of the two strands, likely sets up a proper polarity of template strand binding prior to the association with the replisome. Our search for crystals of the MtDnaG RPD in complex with DNA of different structures, DNA-RNA hybrids, nucleotides and divalent metal ions yielded a 2.45 Å resolution crystal structure of a complex of the RPD with DNA that contained an 8-bp double-stranded region and a 6-nt ss 5’-overhang (Table S2). The backbone of a dinucleotide region of the ss portion of this DNA and a Sr2+ ion were well resolved in the active site in one of the two RPD molecules in the asymmetric unit (Fig. 5A, Fig. S5). The bases were poorly resolved, likely due to their conformational mobility; they were built based on their partial electron density. The dinucleotide backbone is oriented perpendicular to the bound DNA strand seen in the previous structure (Fig. S6). The dinucleotide backbone was modeled to fit best into the difference electron density. Although the polarity of dinucleotide was somewhat ambiguous due to only a modest resolution,


18

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Biochemistry

N

A

B

T271

3’ Y233

R221

D352 3’

K232 R147

C

Y143

D355 5’ 5’

Figure 5. The crystal structure of the MtDnaG RPD bound to the 6-nt 5’-ovehang DNA. The ordered dinucleotide region bound in the active site is shown in orange, and the Sr2+ ion is shown as a purple ball. A. An overall view of the structure. B. A zoomed-in view of the RPD-DNA interface. The dinucleotide interacting residues are shown as dark teal sticks. While the DNA backbone was well ordered, the bases were largely disordered modeled based on their partial electron density.

it appears to be opposite to the bound DNA strand in the previous structure. This opposite polarity is also supported by residual electron density for the backbone of the ds region of this DNA substrate (not included in the final model), bound similarly to the ds DNA in the previous structure (Fig. S7). Thus, the dinucleotide appears to be a part of the ss overhang region of this DNA (residual discontinuous electron density for the rest of this region is also present). Possibly due to the lack of base-specific interactions with the protein, DNA may be bound in different registers to different DnaG molecules in the crystal, resulting in poor residual or discontinuous electron density. The opposite polarities of the bound DNA strand in the previous structure and the dinucleotide in this structure support the model that the former DNA strand mimics the template, whereas the dinucleotide is a mimic of the primer. The dinucleotide is held in the active site by salt bridges and hydrogen bonds with the N-terminal subdomain and by Sr2+ mediated salt bridges with the acidic residues of the TOPRIM subdomain (Fig. 5B). Specifically, both phosphate groups interact with Arg147 and Arg221. Additionally, the phosphate of the nt on the


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Page 20 of 38

3’-side interacts weakly with Lys232 and is at a proper hydrogen bonding distance to the hydroxyl groups of Tyr143, Tyr233 and Thr271. This phosphate is also bound, through the Sr2+ ion, to Asp352 and Asp355. The location of this phosphate group is the same as that of the sulfate bound to the active site in the structure of the free MtDnaG RPD (Fig. 2B). The large number of contacts of this phosphate group with the protein suggests that the positioning of the nt on the 3’-side is highly mechanistically relevant. The contacting basic and the acidic residues are conserved in bacteria and T7 (Fig. S1). These residues have been previously demonstrated to be essential for catalytic activity of E. coli,4, 28, 30 Mt

10

and T7

19, 31

primases. The direct and

extensive coordination of the DNA backbone of this dinucleotide with the key catalytic residues strongly suggests that this dinucleotide mimics a 3’-region of the primer. We superimposed this structure with the structure of the RPD of S. aureus DnaG in complex with an ATP and Mn2+

6

(Fig. 6). Remarkably, the 3’-OH group of the dinucleotide in our

structure is positioned 2 Å away from the phosphorus of the Pα phosphate group of the ATP.

E268

Y233

D272 α

3’

1 D319 T271

2

3

R221 K232

Y143 D352 R147 5’

D355 5’

Figure 6. Superimposition of the dinucleotide (this study; PDB ID: 5W36) with ATP (PDB ID: 4EDG) in the active site of MtDnaG. The Sr2+ ion is shown as a purple ball and the Mn2+ ions coordinating the ATP are shown as chartreuse balls. The dinucleotide, ATP and metal bound residues are shown as dark teal sticks.


20

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Biochemistry

This observation suggests that the dinucleotide mimics the 3’-terminus of the primer positioned for the nucleophilic attack and is consistent with the proposal that the bound ATP is a mimic of the incoming nucleotide. Minor steric overlap between the triphosphate moiety of the ATP and the dinucleotide backbone suggests that the 3’-region of the primer and the triphosphate are somewhat shifted from the positions required for nucleotide transfer. A small movement would allow the 3’-OH to coordinate to metal ion 1 (designated as A in ref. 6) to be activated for nucleophilic attack either directly or through a bound water molecule. As reported previously, the acidic residues coordinating the Mn2+ ions to the ATP are conserved and essential for catalysis in E. coli 4, 28, 30 and T7 19, 31 primases. We also previously showed that the Glu268Gln mutant of MtDnaG was catalytically inactive.10 We examined the environment of the 3’-OH for a general base that could activate this group for the nucleophilic attack. The hydroxyl group of Tyr233 is the only protein residue candidate for a catalytic base. Tyr233 is universally conserved in bacterial primases, but not in T7 primase, where a Phe residue is found in the structurally analogous position.19 The nucleotide transfer activity of the Tyr233Phe mutant of MtDnaG was similar to the wild-type enzyme (Fig. 4C), excluding this residue from its role as a general base and indirectly indicating that a water molecule activated by the divalent metal ion acts as a general base.

DISCUSSION

Crystal structures of the RPD of MtDnaG in complexes with various DNA provide the first glimpse of noncovalent DnaG-type primase-DNA complexes. These structures complement prior biochemical and structural data and fill critical gaps in our understanding of DNA replication in bacteria. The crystal structure of the RPD-ds DNA complex reveals a key site for DnaG


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Page 22 of 38

interaction with ds/ss DNA, located on the N-terminal subdomain of the RPD. The comparable affinity of the RPD to ss and ds DNA and its dominant contribution to DNA binding in the context of the full-length MtDnaG suggest that this site is used by DnaG to slide along ds DNA when idle, to locate the replisome. This binding, to only one of the two strands, could also help engage the template strand with a proper polarity, prior to the association with the replisome. The other crystal structure reveals opposite polarity of the ss dinucleotide to that of the aforementioned DNA strand, and strongly suggests that the DNA strand in the first structure is a mimic of the template strand, whereas the ss dinucleotide is a mimic of the primer. Combined with the structure of the S. aureus DnaG RPD- ATP complex,6 our structure of a dinucleotide in the active site sheds light on how the primer is bound in the active site and how it is extended. We used these structures to generate a model of the RPD MtDnaG bound to the primed template. This complex was then superimposed onto the RPD of the T7 primase closest to the lagging strand T7 DNA polymerase in the recent cryo-EM structure of the T7 replisome,22 to yield a model of the T7 replisome on the primed lagging strand (Fig. 7). The ss DNA in the active site of the DNA polymerase was modeled by superimposing the crystal structure of the T7 DNA polymerase initiation complex.32 This model shows that as the template strand exits the binding site on the primase on the 5’-side, it enters the active site of the DNA polymerase in a proper polarity. This model also shows that the primed region of the template strand in the DnaG active site (built unambiguously to satisfy Watson-Crick base pairing with the primer mimic) is solvent exposed and does not interact with the same “active” RPD. A natural candidate for sequence-specific template binding and its stabilization in the primase active site is the ZBD of the same (in cis) or the adjacent (in trans; in yellow in Fig. 7) primase. The ZBD has been demonstrated to play a key role in template recognition, priming and primer hand-off in to the


22

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Biochemistry

Figure 7. A hybrid T7-MtDnaG model of a lagging strand replisome. A. An MtDnaG RPD (in pale yellow) is modeled in place of the T7 primase RPD domain adjacent to the lagging strand T7 DNA polymerase-thioredoxin complex (in dark gray). The lagging strand (in orange) and the primer (in red) were modeled based on the crystal structures of the MtDnaG RPD-DNA complexes in this study. The lagging strand emerging from the helicase ring is modeled by a dashed line. The RPD and the ZBD of the adjacent primase, interacting in trans with the primertemplate hybrid, are labeled RPD’ and ZBD’ and shown in light gray and yellow, respectively. The T7 helicase is shown in blue. B. A simplified cartoon schematic of the structural model shown in panel A.

polymerase in bacteria and T7.3,

19, 32-35

Geometrically, activation either in cis or in trans is

possible in this model, given a sufficiently long linker between the RPD and the ZBD, as in T7 primase.19 In fact, in T7, initial dinucleotide pppAC synthesis can be carried out in cis, while its extension to a tetranucleotide that is handed over to the DNA polymerase occurs in trans.35-37 However, the action of the ZBD in trans in bacteria is strongly favored by our and previous structural and biochemical data.3 We showed here that the ZBD was required for the primase activity of MtDnaG and that the ZBD rescued the activity of the RPD in trans. Activation in cis would be impossible for A. aeolicus DnaG, where the linker is virtually absent and the ZBD is bound to the RPD far from the active site.3 Modeling the ZBD-primer-template hybrid interaction in trans (Fig. 7) required only a ~45° rotation of the long linker between the RPD and


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Page 24 of 38

the ZBD, starting from the conformation of the ZBD relative to the RPD in the T7 primase structure.19 Such rotation placed the ZBD (in yellow in Fig. 7) in the major groove of the RNADNA hybrid. The orientation of the primer mimic implies that the primer-template hybrid is extruded outward from the primase-helicase ring. This geometry provides an explanation for how the primer length and the primer hand-off to the DNA polymerase may occur. The ZBD would stay bound to the initiation site (due to sequence-specific interactions) and would move radially away from the replisome ring as the primer is elongated by the primase. This movement is limited by the covalent connection of the ZBD with its RPD and would limit the primer length. In this regard, the ZBD is reminiscent of a σ subunit of bacterial RNA polymerase, where this subunit is required for promoter sequence-specific recognition and binding in transcription initiation, while also controlling the escape of the polymerase from the promoter into the elongation mode. As the ZBD moves away from the active site, binding of the DNA in the active site would be destabilized. The primer-template hybrid would then eventually disengage from the RPD and slide to the active site of the DNA polymerase, delivering the 3’-OH for the processive DNA synthesis. The ZBD may stay bound to the initiation site during this hand-off process if only to provide thermodynamic stability of the hybrid, consistent with the observation that the ZBD alone is sufficient to stimulate primer elongation by T7 polymerase.19 CONCLUSION In conclusion, the first crystal structures of noncovalent DnaG-type primase-DNA complexes described here reveal the structure of Mycobacterium tuberculosis primase DnaG and unambiguously establish template binding polarity to an interface that we have validated by


24

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Biochemistry

functional studies. When integrated with prior biochemical and structural data, these structures provide critical insights into replisome organization in bacteria and bacteriophages. Future studies are aimed at testing this replisome model and the mechanistic proposals. ASSOCIATED CONTENT Supporting Information. Supporting Tables and Figures are presented in the supporting information. Tables S1 and S2 contain crystallographic statistics, Table S2 contains primers used to generate mutants, Figure S1 shows multiple sequence alignment of DnaG enzymes from different bacteria, Figure S2 shows the structural comparison of bacterial and bacteriophage primases. The electron density maps for bound DNA are shown in Figs. S3 and S5. The packing of the second MtDnaG monomer in the crystal is shown in Fig. S4. The model of the DNA arrangement in the functioning DnaG based on the observed DNA is in Fig. S6. Fig. S7 shows the structural rationale for the assignment of DNA polarity of the DNA observed to be bound in the active site. These files are available free of charge (PDF).

AUTHOR INFORMATION

Corresponding Author * [email protected] Author Contributions C.H. performed protein expression and purification, X-ray crystallographic experiments, structure determination and refinement, DNA binding and primase activity assays and analyzed the assay data. T.B. cloned MtDnaG constructs, purified MtDnaG and its RPD and crystallized the RPD of MtDnaG. O.V.T. , C.H. and T.B. wrote the manuscript.


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Page 26 of 38

ACKNOWLEDGEMENT We thank Drs. Charles Richardson and Arkadiusz Kulczyk for critical reading of this manuscript, the staff of sector SER-CAT of the Advanced Photon Source for assistance with data collection, the University of Kentucky Center for Structural Biology for the support of SERCAT, and Dr. Sylvie Garneau-Tsodikova for assistance with figure preparation. REFERENCES 1.

Bouche, J. P., Zechel, K., and Kornberg, A. (1975) dnaG gene product, a rifampicinresistant RNA polymerase, initiates the conversion of a single-stranded coliphage DNA to its duplex replicative form, J. Biol. Chem. 250, 5995-6001.

2.

Pan, H., and Wigley, D. B. (2000) Structure of the zinc-binding domain of Bacillus stearothermophilus DNA primase, Structure 8, 231-239.

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Corn, J. E., Pease, P. J., Hura, G. L., and Berger, J. M. (2005) Crosstalk between primase subunits can act to regulate primer synthesis in trans, Mol. Cell 20, 391-401.

4.

Keck, J. L., Roche, D. D., Lynch, A. S., and Berger, J. M. (2000) Structure of the RNA polymerase domain of E. coli primase, Science 287, 2482-2486.

5.

Podobnik, M., McInerney, P., O'Donnell, M., and Kuriyan, J. (2000) A TOPRIM domain in the crystal structure of the catalytic core of Escherichia coli primase confirms a structural link to DNA topoisomerases, J. Mol. Biol. 300, 353-362.

6.

Rymer, R. U., Solorio, F. A., Tehranchi, A. K., Chu, C., Corn, J. E., Keck, J. L., Wang, J. D., and Berger, J. M. (2012) Binding mechanism of metalNTP substrates and stringentresponse alarmones to bacterial DnaG-type primases, Structure 20, 1478-1489.


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Agarwal, A., Louise-May, S., Thanassi, J. A., Podos, S. D., Cheng, J., Thoma, C., Liu, C., Wiles, J. A., Nelson, D. M., Phadke, A. S., Bradbury, B. J., Deshpande, M. S., and Pucci, M. J. (2007) Small molecule inhibitors of E. coli primase, a novel bacterial target, Bioorg. Med. Chem. Lett. 17, 2807-2810.

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Biswas, T., Green, K. D., Garneau-Tsodikova, S., and Tsodikov, O. V. (2013) Discovery of inhibitors of Bacillus anthracis primase DnaG, Biochemistry 52, 6905-6910.

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Biswas, T., Resto-Roldan, E., Sawyer, S. K., Artsimovitch, I., and Tsodikov, O. V. (2013) A novel non-radioactive primase-pyrophosphatase activity assay and its application to the discovery of inhibitors of Mycobacterium tuberculosis primase DnaG, Nucleic Acids Res. 41, e56.

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Pang, A. H., Garneau-Tsodikova, S., and Tsodikov, O. V. (2017) In Vitro Assays to Identify Antibiotics Targeting DNA Metabolism, Methods Mol. Biol. 1520, 175-200.

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Kuron, A., Korycka-Machala, M., Brzostek, A., Nowosielski, M., Doherty, A., Dziadek, B., and Dziadek, J. (2014) Evaluation of DNA primase DnaG as a potential target for antibiotics, Antimicrob. Agents Chemother. 58, 1699-1706.

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Gajadeera, C., Willby, M. J., Green, K. D., Shaul, P., Fridman, M., Garneau-Tsodikova, S., Posey, J. E., and Tsodikov, O. V. (2015) Antimycobacterial activity of DNA intercalator inhibitors of Mycobacterium tuberculosis primase DnaG, J. Antibiot. 68, 153157.

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Richardson, C. C. (2015) It seems like only yesterday, Annu. Rev. Biochem. 84, 1-34.


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Ilic, S., Akabayov, S. R., Arthanari, H., Wagner, G., Richardson, C. C., and Akabayov, B. (2016) Identification of DNA primase inhibitors via a combined fragment-based and virtual screening, Sci. Rep. 6, 36322.

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Choi, S. R., Larson, M. A., Hinrichs, S. H., and Narayanasamy, P. (2016) Development of potential broad spectrum antimicrobials using C2-symmetric 9-fluorenone alkyl amine, Bioorg. Med. Chem. Lett. 26, 1997-1999.

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Robinson, A., Causer, R. J., and Dixon, N. E. (2012) Architecture and conservation of the bacterial DNA replication machinery, an underexploited drug target, Curr. Drug Targets 13, 352-372.

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Bailey, S., Eliason, W. K., and Steitz, T. A. (2007) Structure of hexameric DnaB helicase and its complex with a domain of DnaG primase, Science 318, 459-463.

19.

Kato, M., Ito, T., Wagner, G., Richardson, C. C., and Ellenberger, T. (2003) Modular architecture of the bacteriophage T7 primase couples RNA primer synthesis to DNA synthesis, Mol. Cell 11, 1349-1360.

20.

Toth, E. A., Li, Y., Sawaya, M. R., Cheng, Y., and Ellenberger, T. (2003) The crystal structure of the bifunctional primase-helicase of bacteriophage T7, Mol. Cell 12, 11131123.

21.

Corn, J. E., Pelton, J. G., and Berger, J. M. (2008) Identification of a DNA primase template tracking site redefines the geometry of primer synthesis, Nat. Struct. Mol. Biol. 15, 163-169.

22.

Kulczyk, A. W., Moeller, A., Meyer, P., Sliz, P., and Richardson, C. C. (2017) Cryo-EM structure of the replisome reveals multiple interactions coordinating DNA synthesis, Proc. Natl. Acad. Sci. U. S. A. 114, 1848-1856.


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23.

Biswas, T., and Tsodikov, O. V. (2008) Hexameric ring structure of the N-terminal domain of Mycobacterium tuberculosis DnaB helicase, Febs J 275, 3064-3071.

24.

Otwinowski, Z., Minor, W., and et al. (1997) Processing of X-ray diffraction data collected in oscillation mode, Methods Enzymol 276, 307-326.

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McCoy, A. J., Grosse-Kunstleve, R. W., Adams, P. D., Winn, M. D., Storoni, L. C., and Read, R. J. (2007) Phaser crystallographic software, J Appl Crystallogr 40, 658-674.

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Emsley, P., Lohkamp, B., Scott, W. G., and Cowtan, K. (2010) Features and development of Coot, Acta Crystallogr D Biol Crystallogr 66, 486-501.

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Murshudov, G. N., Vagin, A. A., and Dodson, E. J. (1997) Refinement of macromolecular structures by the maximum-likelihood method, Acta Crystallogr D Biol Crystallogr 53, 240-255.

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Rodina, A., and Godson, G. N. (2006) Role of conserved amino acids in the catalytic activity of Escherichia coli primase, J Bacteriol 188, 3614-3621.

29.

Tsodikov, O. V., Enzlin, J. H., Scharer, O. D., and Ellenberger, T. (2005) Crystal structure and DNA binding functions of ERCC1, a subunit of the DNA structure-specific endonuclease XPF-ERCC1, Proc Natl Acad Sci U S A 102, 11236-11241.

30.

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Lee, S. J., and Richardson, C. C. (2005) Acidic residues in the nucleotide-binding site of the bacteriophage T7 DNA primase, J Biol Chem 280, 26984-26991.


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33.

Kulczyk, A. W., and Richardson, C. C. (2012) Molecular interactions in the priming complex of bacteriophage T7, Proc Natl Acad Sci U S A 109, 9408-9413.

34.

Lee, S. J., Zhu, B., Hamdan, S. M., and Richardson, C. C. (2010) Mechanism of sequence-specific template binding by the DNA primase of bacteriophage T7, Nucleic Acids Res 38, 4372-4383.

35.

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Kulczyk, A. W., and Richardson, C. C. (2016) The Replication System of Bacteriophage T7, Enzymes 39, 89-136.

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Biochemistry

For Table of Contents Use Only

Structures of the catalytic domain of bacterial primase DnaG in complexes with DNA provide insight into key priming events Caixia Hou,a Tapan Biswas,b and Oleg V. Tsodikova,*

DNA

DnaG


31

Biochemistry

A 0.35


0.30 0.25 0.20 0.15 0.10 0.05

ss DNA, Kd = 0.18 ± 0.03 μM ds DNA, K d = 0.33 ± 0.05 μM 0

500

B 0.35

1000 1500 [full-length MtDnaG], nM

2000

2500


0.30 0.25 0.20 0.15 0.10 0.05 C

ss DNA, Kd = 0.83 ± 0.16 μM ds DNA, K d = 0.50 ± 0.08 μM 0

500

1000 1500 [MtDnaG RPD], nM

2000

2500

100

80


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 38 Figure 1

60

40

20

0 MtDnaG DNA

f.l. wt

ACS Plus Environment f.l. wtParagon RPD ZBD RPD+ZBD ZBD-RPD

Page 33 of 38 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

Biochemistry

Figure 2

A

N

C

B

C Y233 β2

R199 K232

R221

R190 β3

Y143 R147


M229

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

A

Page 34 of 38

Figure 3

B

3’

5’

C

R193 2 5

N

E230 M229

3

4

1

R190 H194 M197 W166


Page 35 of 38

Biochemistry A 0.28


0.26

wild-type

ds DNA

0.24

R199A

0.22 0.20

R190A

0.18 0.16 0.14 0.12 0.10 0.0


B 0.30

C

0.5


2.0

2.5 wild-type

ss DNA

0.25

R199A

0.20 R190A 0.15

0.10

0.05 0.0

0.5


100

80


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

Figure 4

60

40

20

0 MtDnaG

ACS Paragon Plus Environment Y233F wild-type R199A R190A

2.0

2.5

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

N

A

Page 36 of 38

Figure 5

B T271

3’

Y233

R221

D352 3’

K232 R147

C

D355 5’


Y143 5’

Page 37 of 38 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

Biochemistry

Figure 6

E268

Y233

D272 α

D319 T271

2

3’

1

R221

3 Y143

K232

D352 R147

5’

D355 5’


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

A

Page 38 of 38

B 5’ lagging strand DNA pol 5’ DNA pol template RPD’ template

RPD’

3’

primer

primer

ZBD’

ZBD’

RPD helicase


RPD

Structures of the Catalytic Domain of Bacterial ... - ACS Publications

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