An Interaction Between DNA Polymerase and Helicase Is Essential for the High Processivity of the Bacteriophage T7 Replisome

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An Interaction between DNA Polymerase and Helicase Is Essential for the High Processivity of the Bacteriophage T7 Replisome

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Citation Kulczyk, Arkadiusz W., Barak Akabayov, Seung-Joo Lee, Mihnea Bostina, Steven A. Berkowitz, and Charles C. Richardson. 2012. “An Interaction between DNA Polymerase and Helicase Is Essential for the High Processivity of the Bacteriophage T7 Replisome.” Journal of Biological Chemistry 287 (46): 39050–60. https://doi.org/10.1074/ jbc.m112.410647.

Citable link http://nrs.harvard.edu/urn-3:HUL.InstRepos:41483402

Terms of Use This article was downloaded from Harvard University’s DASH repository, and is made available under the terms and conditions applicable to Other Posted Material, as set forth at http:// nrs.harvard.edu/urn-3:HUL.InstRepos:dash.current.terms-of- use#LAA THE JOURNAL OF BIOLOGICAL CHEMISTRY VOL. 287, NO. 46, pp. 39050–39060, November 9, 2012 © 2012 by The American Society for Biochemistry and Molecular Biology, Inc. Published in the U.S.A.

An Interaction between DNA Polymerase and Helicase Is Essential for the High Processivity of the Bacteriophage T7 Replisome*□S Received for publication, August 15, 2012, and in revised form, September 12, 2012 Published, JBC Papers in Press, September 13, 2012, DOI 10.1074/jbc.M112.410647 Arkadiusz W. Kulczyk‡, Barak Akabayov‡, Seung-Joo Lee‡, Mihnea Bostina§, Steven A. Berkowitz¶, and Charles C. Richardson‡1 From the ‡Department of Biological Chemistry and Molecular Pharmacology, Harvard University Medical School, Boston, Massachusetts 02115, the §Facility for Electron Microscopy Research, McGill University, Montreal, Quebec H3A 2B2, Canada, and ¶Biogen Idec, Inc., Cambridge, Massachusetts 02142

Background: Interactions of DNA polymerase and DNA helicase are crucial in DNA synthesis. Results: Two distinct interactions are involved in formation of the DNA polymerase/DNA helicase complex. Conclusion: The multiple interactions between DNA polymerase and DNA helicase account for the high processivity of leading strand synthesis. Significance: Understanding of the replication process in bacteriophage T7 facilitates studies in more complex systems. Downloaded from

Synthesis of the leading DNA strand requires the coordinated The primase contains the N-terminal zinc-binding domain activity of DNA polymerase and DNA helicase, whereas synthe- (ZBD)2 connected to the RNA polymerase domain (RPD). The sis of the lagging strand involves interactions of these proteins helicase domain uses the energy derived from the hydrolysis of with DNA primase. We present the first structural model of a dTTP to translocate unidirectionally on ssDNA (3). Upon bacteriophage T7 DNA helicase-DNA polymerase complex encountering dsDNA, it unwinds the DNA. The primase http://www.jbc.org/ using a combination of small angle x-ray scattering, single-mol- domain recognizes specific sequences on the lagging strand and ecule, and biochemical methods. We propose that the protein- synthesizes tetraribonucleotides that are used as primers for protein interface stabilizing the leading strand synthesis the lagging strand DNA polymerase (4). T7 DNA polymerase involves two distinct interactions: a stable binding of the heli- (gp5) contains an N-terminal proofreading exonuclease do- case to the palm domain of the polymerase and an electrostatic main and a C-terminal polymerase domain. gp5 forms a one to by guest on October 5, 2019 binding of the carboxyl-terminal tail of the helicase to a basic one complex with its processivity factor thioredoxin (trx) (5). patch on the polymerase. DNA primase facilitates binding of The crystal structure of the gp5 and trx complex (gp5/trx) DNA helicase to ssDNA and contributes to formation of the bound to a primer/template revealed that trx binds to a 76- DNA helicase-DNA polymerase complex by stabilizing DNA amino acid loop inserted into the thumb subdomain (thiore- helicase. doxin-binding domain) (6). Coordination of leading and lagging strand DNA synthesis, the formation and resolution of a replication loop, and the Because of its simplicity, bacteriophage T7 provides a model exchange of DNA polymerases implies that the four proteins system for the molecular interactions during DNA replication interact with one another. A number of these interactions have (1). Only four proteins are required for the basic phage repli- been described (see Fig. 1A). The binding of trx to gp5 alters the some, yet the reconstituted replisome recapitulates all the key configuration of the thioredoxin-binding domain such that it features of more complex systems (see Fig. 1A). The four pro- can clasp the DNA within the DNA-binding crevice (7). This teins are: DNA polymerase and its processivity factor, Escheri- interaction also remodels the thioredoxin-binding domain to chia coli thioredoxin, DNA primase-helicase, and ssDNA-bind- create two solvent-exposed basic loops (exchange patch) to ing protein. The limited number of proteins has made possible: which gp4 and gp2.5 can bind (8). Interactions of the C-termi- (i) the determination of their crystal structures, (ii) reconstitu- nal tail of gp4 with the exchange patch of gp5 increase the pro- tion of a replisome that mediates coordinated DNA synthesis, cessivity of the replisome from 5 to Ͼ17 kb by capturing gp5/trx and (iii) visualization of active replisomes by single-molecule that transiently dissociates (9, 10). Another electrostatic inter- techniques. action between the 17 C-terminal amino acid tail of gp4 and a The protein product of T7 gene 4 (gp4) provides helicase and basic patch of gp5 (loading patch) is essential for the loading of primase activities. The helicase resides within the C-terminal gp5/trx onto the replication fork (11). half of the protein and the primase in the N-terminal half (2). There is a third unidentified interaction that does not involve the C-terminal tail of gp4. gp5/trx has a processivity of only 0.8 * This work was supported, in whole or in part, by National Institutes of Health Grant GM-54397 (to C. C. R.). □S This article contains supplemental Tables S1–S3 and Figs. S1 and S2. 2 The abbreviations used are: ZBD, zinc-binding domain; RPD, RNA polymer- 1 To whom correspondence should be addressed: Harvard University Medical ase domain; SAXS, small angle x-ray scattering; trx, thioredoxin; AUC, ana- School, 240 Longwood Ave., Boston, MA 02115. Tel.: 617-432-1864; Fax: lytical ultracentrifugation; SPR, surface plasmon resonance; ssDNA, single- 617-432-3362; E-mail: [email protected]. stranded DNA; LMW, low molecular weight; Ru, resonance unit.

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kb on ssDNA but a processivity of Ͼ17 kb during leading strand containing 1 mM dGTP and 10 ␮M ddATP. gp4 variants were synthesis when coupled to gp4 (12). A deletion of the C-termi- injected in buffer consisting of 0.1 mM ATP and 2 mM dGTP. nal tail of gp4 reduces the processivity to 5 kb. Therefore, a The flow cell blocked with biotin was used to measure the non- third, uncharacterized interaction of gp4 and gp5/trx is essen- specific interactions and bulk refractive index. The data were tial for the high processivity of 5 kb observed during DNA syn- analyzed using the software BIAEVAL 4.1. thesis in the absence of gp5/trx exchange. In the current study, Sedimentation Velocity Analytical Ultracentrifugation— we dissect the interactions that stabilize the complex of gp4 and AUC analysis of proteins was conducted using Beckman gp5/trx during leading strand synthesis. Coulter ProteomeLab XL-I centrifuge. The experiments were conducted at 20 °C using an eight-hole (An-50 Ti) rotor. Prior EXPERIMENTAL PROCEDURES to AUC analysis, the samples were buffer-exchanged and con- Protein Expression and Biochemical Assays—Proteins were centrated using centrifugation cartridges with 10,000 molecu- overexpressed and purified as described previously: WT gp4 and lar mass cutoff membranes into buffer consisting of 20 mM gp4⌬ZBD (13), gp4 ⌬C (14), gp4⌬helicase, gp4⌬C⌬primase, gp5, Tris-HCl, pH 7.5, 200 mM NaCl, 2 mM MgCl2, and 5 mM dTTP. and trx (15). In the SPR, EM, and small angle x-ray scattering The samples were then diluted to their target concentration (SAXS) experiments, we use a gp5/trx in which two amino acids using the same buffer used in conduct buffer exchange. All (Asp-5 and Glu-7) in the exonuclease domain of gp5 are replaced boundary sedimentation velocity runs were conducted at rotor Ј Ј with Ala; the 3 to 5 proofreading exonuclease activity is reduced speed of 40,000 or 45,000 rpm. AUC cells used for these exper- 6 by a factor of 10 (6). The biochemical assays have been described iments were assembled with 12-mm synthetic boundary double in detail earlier: DNA binding and oligomerization assays (16),

sector charcoal-filled epon centerpieces and sapphire windows. Downloaded from unwinding assay (17), and minicircle assay (18). The reference side of the double sector AUC cell was always ␭ Single-molecule Measurements—Phage DNA (48.5 kb) filled with an excess of 20 ␮l of buffer solution in comparison molecules containing a replication fork were attached with the with the samples sector. AUC cells were then placed into the Ј 5 -end of one strand to the glass surface of a flow cell via biotin- rotor and accelerated to 15,000–20,000 rpm to transfer the streptavidin link. The 3Ј-end of the same strand was attached to excess buffer in the reference sector over to the samples sector, a 2.8-␮m paramagnetic bead (Dynal) via digoxigenin-anti- http://www.jbc.org/ so the radial distance of the meniscus from the center of rota- digoxigenin. To prevent nonspecific interactions between the tion for both the sample and reference sectors matched. Once beads and the surface, a 1-pN magnetic force was applied buffer was transferred, the rotor was stopped, and the cells were upward by positioning a permanent magnet above the flow cell. removed and gently mixed by rotating the cell a number of The beads were imaged with a CCD camera with a time reso- times. The rotor, containing the cells, was then placed back into

lution of 500 ms, and the centers of their positions for every by guest on October 5, 2019 the centrifuge where it was allowed to temperature equilibrate acquisition time point were determined by particle-tracking for 1.5–2 h before the run was started. Data acquisition was software. Bead-bound and surface-tethered DNA were prein- carried out every 5 min using the Raleigh interferometer. Data cubated with 300 nM of different variants of gp4 (monomeric analysis was conducted using the c(s) method in Peter Shuck’s concentration), 100 nM gp5/trx in buffer: 40 mM Tris-HCl, pH program SEDFIT (19) using a regularization confidence level of 7.5, 50 mM potassium glutamate, 2 mM EDTA, 0.1 mg/ml BSA, 0.68 and data radial spacing of 0.003 cm. 10 mM DTT, consisting of 600 ␮M each of dATP, dTTP, dCTP, and dGTP. Next, the flow cell was washed with replication Electron Microscopy and Image Processing—Uranyl formate buffer with dNTPs. DNA synthesis was initiated by introducing samples were prepared as described (20). Images were collected at a Tecnai F-20 electron microscope (FEI, Eindhoven, The replication buffer with dNTPs and 10 mM MgCl2. For data analysis, after particle tracking, the traces were corrected for resid- Netherlands) at an acceleration voltage of 200 kV with a mag- ϫ ual instabilities in the flow by subtracting traces corresponding nification of 50,000 corresponding to 2.25 pixel size, at a defo- ␮ to tethers that were not enzymatically altered. Bead displace- cus in range of 0.5–1.4 m. Particle images were selected for each ϳ ments were converted into numbers of nucleotides synthesized sample using the software Signature (21). A stack of 5000 images using the known length difference between ssDNA and dsDNA were windowed into 128-pixel boxes. Images analysis was done at our experimental conditions (12). using SPARX (22). Several rounds of K-means classification and Surface Plasmon Resonance—SPR analysis was performed multireference were applied. using a Biacore 3000 instrument (Uppsala, Sweden). Binding Small Angle X-ray Scattering—The sample for SAXS mea- experiments between gp5/trx and gp4 variants in the presence surements was prepared by mixing 30 ␮M gp4⌬primase bound of primer/template were performed as previously described (8). to a 15-mer oligonucleotide (GGGTCA10) in the presence of The template (5Ј-CCCCCTTGGCACTGGCCGTCGTTTTC- ␤,␥-methylene dTTP with 5 ␮M gp5/trx bound to the previ- ACGTT-biotin-3Ј) and the primer (5Ј-CGTGAAAACGACG- ously annealed and purified 5.2 ␮M primer/template DNA (5Ј- GCCAGTGCCA-3Ј) strands were annealed, and 350 RU of a CGAAAACGACGGCCAGTGCCA-3Ј/5Ј-CCCCTTGGCACTG- primer/template were coupled to the SA chip. As a control, one GCCGTCGTTTTCG-3Ј) in the presence of 0.1 mM ddATP, and flow cell on the chip was blocked with free biotin. Binding stud- 0.1 mM dGTP in the following buffer: 50 mM potassium phos- ies were carried out in buffer containing 20 mM HEPES, pH 7.5, phate, pH 7.5, 10% glycerol, and 1 mM DTT. The measurements

200 mM potassium glutamate, 10 mM MgCl2,5mM DTT, 1% were performed at the National Synchrotron Light Source (w/v) glycerol, and 0.5 mM dGTP at a flow rate of 10 ␮l/min. (Upton, NY) on Beamline X-9. The magnitude of the scattering gp5/trx was injected at the concentration of 200 nM in buffer vector (q) is defined as follows,

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4␲sin␪ ϭ q ␭ (Eq. 1)

where 2␪ is the scattering angle. The experimental SAXS data for the sample showed a linear behavior in the low q, Guinier region, indicating that the leading strand complex did not undergo aggregation. Radii of gyra- Ͻ tion (Rg) were derived from data in the qRg 1.3 region using the Guinier approximation.

Ϫ 2 2 Rgq I͑q͒ ϭ I͑0͒exp (Eq. 2) 3

The scattering curve reflects structural characteristics in recip- rocal space. The software GNOM was used to translate the scattering profiles into real space by Fourier transformation, resulting in the pairwise-distance distribution function (p(r)). The p(r) function is proportional to the particle electron den-

sity and reflects the distances between pairs of scattering points Downloaded from within the macromolecule, permitting determination of the

maximum intramolecular diameter (Dmax). To obtain a reliable quantification of the maximum diameter of the examined particles (D ), we utilized the methods described earlier (23), FIGURE 1. The replisome of bacteriophage T7 and variants of gp4. A, the max replisome of bacteriophage T7 contains DNA polymerase (gp5) and its pro- which was incorporated into our in-house scripts. In this cessivity factor, E. coli thioredoxin (trx), DNA primase-helicase (gp4), and http://www.jbc.org/ method, Dmax was set as a variable in the range of 100–300 Å ssDNA-binding protein (gp2.5). gp4 unwinds dsDNA and generates two and varied in increments of 10 Å. Evaluation parameters ssDNA templates for the leading and lagging strand gp5/trx. gp2.5 coats the lagging strand ssDNA. The primase domain of gp4 catalyzes the synthesis of described by Lipfert et al. (23) were analyzed, and the values of oligoribonucleotides (green) that function as primers for the initiation of each Okazaki fragment. B, gp4 consists of two major domains connected by a flex- Dmax that showed the best overall behavior and GNOM statistics were chosen for the subsequent structure reconstruction ible linker: the C-terminal helicase and the N-terminal primase. The primase domain contains two subdomains: the N-terminal ZBD and the RPD that are by guest on October 5, 2019 (supplemental Fig. S2). also connected by a flexible linker. The C-terminal 17 amino acids of the heli- Three-dimensional Structure Reconstruction—The three-di- case domain are unstructured. The positions of residues important in the construction of the various forms of g4 are indicated. We constructed five mensional shape of the gp4⌬primase-ssDNA in complex with truncated proteins: gp4⌬helicase (residues 1–271) lacks the helicase domain gp5/trx-primer/template was obtained from the experimental but contains the flexible linker connecting the RPD with the helicase domain, gp4⌬C (residues 1–549) lacks the C-terminal 17 amino acids, gp4⌬ZBD (resi- scattering data using the software GASBOR (24). All rigid body dues 64–566) lacks the N-terminal ZBD but includes a part of the flexible analysis programs employ amplitudes from the computer pro- linker that connects the ZBD and RPD, gp4⌬primase (residues 241–566) lacks gram CRYSOL that also explicitly simulates the solvation shell both the N-terminal ZBD and RPD but contains the flexible linker that connects the RPD and the helicase domain, and gp4⌬C⌬primase consists of the bya3Ålayer on the surface. GASBOR performs ab initio helicase domain and the flexible linker connecting helicase with the RPD but shape reconstruction that represents the molecular shape as is lacking both the primase domain and the C-terminal acidic tail. a closely packed sphere assembly within a search volume defined by the maximum particle dimensions (Dmax) chosen Physical Interactions between the DNA Helicase Domain of using the search script mentioned above. Ten low resolution gp4 and gp5/trx Stabilize the Leading Strand Complex—During models were averaged using the program DAMAVER to DNA synthesis gp5/trx extends the leading strand, whereas the yield an averaged model representing the general structural gp4 hexamer encircles the lagging ssDNA. Terminating the features of each reconstruction. primer with a ddNMP and providing the next dNTP specified by the template yields a gp5/trx-primer/template complex that RESULTS mimics ongoing leading strand synthesis, and its structure was Genetically Altered Variants of DNA Primase-Helicase—To solved to a 2.2 Å resolution (6). gp4 and gp5/trx form a stable determine the contributions of the individual domains of gp4 to complex under these conditions (8). Therefore, to dissect the interactions with gp5/trx, the WT gp4 was genetically altered interactions involved in formation of the leading strand repli- to create five gp4 variants (Fig. 1B). gp4⌬helicase lacks the cation complex, we have examined the ability of gp4 variants to entire helicase domain. gp4⌬C lacks the 17 C-terminal amino bind to gp5/trx under these conditions using SPR. Fig. 2A acids. gp4⌬ZBD lacks the N-terminal 63 residues, a deletion shows a cartoon of gp5/trx locked in a polymerization mode on that removes the ZBD of the primase domain. gp4⌬primase is a a primer/template. more extensive deletion, resulting in removal of the entire pri- The SPR sensorgrams in Fig. 2B show the binding between mase domain. gp4⌬C⌬primase lacks the entire primase gp5/trx-primer/template and the different gp4 variants that domain and the C-terminal amino acids. In the following sec- were subsequently injected into the flow cell. Because the gp4 tion we assess the binding of these variants to gp5/trx. variants differ in mass from the WT gp4, we normalized the

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thesis. In a control, we found no detectable binding of gp4 to the immobilized DNA alone. The ZBD, RPD, and the helicase domain of gp4 are connected by flexible linkers. Therefore removal of these domains does not change the surface and thus does not create an artificial interaction with gp4. Single-molecule Analysis of Leading Strand Synthesis— Single-molecule techniques allow for the quantitative measurement of the rate and processivity of DNA synthesis catalyzed by individual replisomes (12, 26). Fig. 3A shows a schematic rep- resentation of the single-molecule assay we used. Linearized ␭ DNA is modified to form a structure resembling the replication fork at one end. The fork-shaped DNA ligand has biotin on the 3Ј-end of one strand and a digoxigenin on the 5Ј-end of the same strand. The biotinylated end is attached to a coverslip, and the digoxigeninated end is attached to a 2.8-␮m bead. A lami- nar flow of 3 pN is applied, and the resultant drag stretches the molecule of ␭ DNA. At 3 pN the elasticity of DNA is determined by entropic contributions, and ssDNA is shorter than dsDNA FIGURE 2. SPR analysis of binding of gp4 to gp5/trx-primer/template. because of coiling of the ssDNA. Consequently, by measuring

A, cartoon of gp5/trx locked in a polymerization mode on a primer/template. Downloaded from The primer/template consists of a 23-nucleotide primer annealed to a 31-nu- the decrease in length of the DNA molecule, we monitor con- cleotide template. The 3Ј-end of the template DNA is biotinylated to enable version of dsDNA to ssDNA as a result of the leading strand the immobilization to a streptavidin (SA) sensor chip. gp5/trx is injected in synthesis. These measurements are accomplished by imaging buffer containing dGTP and ddATP. ddAMP terminates the primer strand, thus preventing incorporation of the next incoming nucleotide to the primer the bead position as a function of time (Fig. 3A). strand, and dGTP, the next dNTP specified by the template, locks gp5/trx on gp5/trx and gp4 were flowed over the immobilized DNA in

DNA (6). B, 350 RU of a primer/template were coupled to the SA chip. gp5/trx http://www.jbc.org/ was injected in buffer containing dGTP and ddATP. The presence of nucleo- buffer containing dNTPs. After loading onto the DNA, free tides in buffer allows formation of approximately 1500 RU of the stable com- protein was removed by washing the flow cell with buffer. Lead- plex between gp5/trx and the primer/template. WT gp4 and the gp4 variants ing strand synthesis was initiated by flowing buffer containing were subsequently injected in buffer consisting of ATP and dGTP. The number of RU on the y axis reflects the strength of binding between an individual MgCl2. The processivity is determined by measuring the length gp4 variant and gp5/trx-primer/template. The initiation of the protein injec- of each DNA molecule from the beginning to the end of the tions is indicated by arrows. C, a histogram showing the relative binding of gp4 variants. Because of differences of gp4 variants in mass, we multiplied the shortening event. The rate is obtained by fitting each shorten- by guest on October 5, 2019 maximal RU values (Rmax) recorded for each individual gp4 by the ratio of the ing trajectory with linear regression and calculating the slope. molecular weight of the WT protein to that of the altered protein. The results All individual processivity and rate measurements are then of normalization are presented as relative percentages in respect to WT gp4 binding. combined in distributions and fitted with exponential and Gaussian functions, respectively (Fig. 3B). Representative tra-

data by multiplying the maximal RU (Rmax) recorded for gp4 jectories and the averaged values are shown in Fig. 3C. variants by the ratio of the mass of the WT protein to that of the With gp5/trx and WT gp4 synthesis proceeds at a rate of altered protein (Fig. 2C). 112 Ϯ 24 nucleotides/s and a processivity of 16 Ϯ 4 kb. Substi- WT gp4 forms a stable complex as evidenced by a rapid tution of WT gp4 with gp4⌬ZBD results in a 27% decrease in increase in RU after gp4 injection and a slow dissociation from rate and a 38% decrease in processivity, suggesting that the the complex. gp4⌬ZBD and gp4⌬primase bind at 111 Ϯ 2 and deletion of ZBD has a minimal influence on synthesis. Substi- 133 Ϯ 13% of the level observed with WT gp4, respectively. tution of gp4⌬C for WT gp4 results in a 20% decrease in rate Both of these gp4 associate with the complex in a similar man- and a decrease in processivity to 4 kb. We showed previously ner as the WT protein. However, they dissociate at a faster rate. that gp5/trx dissociates from the DNA approximately every 5 The data indicate that the ZBD and RPD contribute to the sta- kb and can either be retained by the C-terminal tail of gp4 or bility of the complex. The binding of gp4⌬helicase lacking the exchange with free gp5/trx in solution (27). No shortening tra- entire helicase domain but containing the primase domain sup- jectories, indicative of no synthesis, were detected with ports this conclusion. gp4⌬helicase displays less than 10% of gp4⌬primase and gp4⌬C⌬primase. the level of binding measured for WT gp4. A similar stabiliza- In the replication system of B. stearothermophilus, the inter- tion effect is observed in the crystal structure of Bacillus stearo- action between the primase (DnaG) and the helicase (DnaB) thermophilus helicase bound to the helicase-binding domain of increases the NTPase and the unwinding activities of DnaB (28) the primase (25). and consequently increases the processivity of replication. The As expected, a deletion of the C-terminal tail of gp4 (gp4⌬C) crystal structure of DnaB bound to helicase-binding domain of decreases the binding of gp4 (68 Ϯ 9% of the level recorded for DnaG shows that the conformation of DnaB hexamer is stabi- the WT protein) (9). To test directly whether DNA helicase is lized by binding of the three helicase-binding domain to the involved in formation of the leading strand complex, we meas- surface of DnaB (25). Analogically, the interaction between the ured binding of gp4⌬C⌬primase. gp4⌬C⌬primase binds at primase and the helicase of gp4 fulfills this role during leading 31 Ϯ 3% of the level observed with WT gp4. Thus the helicase strand synthesis. The lack of activity exhibited by gp4⌬primase domain of gp4 interacts with gp5/trx during leading strand syn- and gp4⌬C⌬primase can result from an inability of the trun-

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FIGURE 3. Single-molecule measurement of leading strand DNA synthesis. A, the experimental design. A phage ␭ DNA molecule (48.5 kb) containing a replication fork is attached to the surface of the flow cell via the 5Ј-end of one strand using biotin-streptavidin interaction. The 3Ј-end of the same strand is attached to a 2.8-␮m paramagnetic bead via digoxigenin-anti-digoxigenin interaction. gp5/trx and gp4 are preassembled at the replication fork in the presence of dNTPs, but in the absence of Mg2ϩ. Free proteins are removed by washing the flow cell with buffer that does not contain proteins. The reaction is initiated by the addition of Mg2ϩ. The conversion of the dsDNA to ssDNA results in shortening the DNA. The position of the bead is recorded and analyzed. B, rate and processivity distributions. All individual single-molecule trajectories for leading strand synthesis were combined and plotted as histograms. The rates and processivities were calculated by fitting the distributions using Gaussian and exponential functions, respectively. C, the calculated rates and processivities of leading strand synthesis obtained with different gp4 and examples of single-molecule trajectories. 22, 31, and 31 single events were used to calculate rates and processivities for WT gp4, gp4⌬ZBD, and gp4⌬C, respectively. No shortening trajectories were observed with gp4⌬primase and gp4⌬C⌬primase. cated proteins to oligomerize, weak ssDNA binding, or a defi- The quantification of samples is presented in supplemental ciency in unwinding of dsDNA. Table S1. The results show little change in species composition Analytical Ultracentrifugation Studies of the Oligomerization as a function of total protein concentration. Plotting the ⌬ of gp4 primase—We used analytical ultracentrifugation to weight-average s (sw) for each sample versus the total protein ⌬ examine the oligomerization properties of gp4 primase and concentration reveals a decreasing relationship of sw with performed sedimentation velocity experiments (AUC) with increasing concentration (supplemental Fig. S1A). The data interference optics. Three samples of gp4⌬primase were ana- indicate the absence of any significant dynamic equilibrium ⌬ lyzed at different concentrations, in buffer containing MgCl2 behavior between any gp4 primase species present. and dTTP. Fig. 4A shows the noise-corrected raw AUC data As a control we characterized WT gp4 at different concen- (upper panel) for the sample at 1 mg/ml and its associated cal- trations. Fig. 4B shows the analogous data calculated for WT culated distributions of sedimentation coefficients (c(s)) plot- gp4 at 1.1 mg/ml. The c(s) plotted versus s for each gp4 sample ted versus sedimentation coefficient (s) expressed in unit of shows the presence of two peaks between 11 and 14 S. Previous Svedberg (S)(lower panel). The plot of c(s) versus s for each analysis of WT gp4 samples by electron microscopy revealed sample shows the presence of three groups of peaks: (i) Ͻ8S the presence of hexameric and heptameric rings with heptam- corresponds to low molecular weight (LMW) oligomers, likely ers varying between 33% (29) and 78% (30). Therefore it is likely monomers, dimers, and trimers; (ii) 8–9 S, most likely the hexa- that these two peaks (the slow and fast migrating peak) corre- meric helicase; and (iii) Ͼ10 S corresponds to high molecular spond to the hexameric and heptameric gp4, respectively. weight (HMW) aggregates. Although LMW and high molecular weight species are also

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FIGURE 4. Sedimentation velocity and electron microscopy studies of gp4⌬primase and WT gp4. A and B, the boundary sedimentation velocity experi- ⌬ ments were conducted at a rotor speed of 45000 rpm. The samples consisted of gp4 primase (1 mg/ml) or WT gp4 (1.1 mg/ml), MgCl2, and dTTP. The upper panels in A and B show respectively the noise corrected raw analytical ultracentrifugation data acquired for gp4⌬primase and WT gp4 at the above concen- http://www.jbc.org/ trations. The lower panels in A and B show the associated deconvoluted c(s) values plotted versus s expressed in Svedberg units (S) for gp4⌬primase and WT gp4, respectively. Plots show the presence of a number of peaks that were arranged into the following three groups: (i) LMW oligomers, likely representing an incomplete assembled protein, i.e., monomers, dimers, and trimers; (ii) material migrating between 8 and9Sinthegp4⌬primase sample and between 11 and 14 S in the WT gp4 sample, both corresponding to the assembled helicase (likely hexameric form for gp4⌬primase, hexameric, and heptameric forms for WT gp4); and (iii) high molecular weight aggregate. Integration of these plots and quantification of subsequent groups of peaks is presented in supplemental Tables S1 and S2 for gp4⌬primase and WT gp4, respectively. C, visualization of gp4⌬primase and WT gp4 by negative staining electron microscopy. gp4⌬primase and WT gp4 were incubated in the buffer containing MgCl and dTTP. After 30 min of incubation at 25 °C, the samples were stained with uranyl

2 by guest on October 5, 2019 formate. The figure shows a representative image of the negatively stained gp4⌬primase. D, representative projection averages of the octameric, heptameric, and hexameric rings obtained by multireference alignment and classification. Images of 5295 particles were selected for WT gp4. For gp4⌬primase we selected 4870 images of particles. Image classification revealed the presence of three different forms of gp4 in both samples: hexamers, heptamers, and octamers. The relative content of each species is specified in a table below the figure.

detected in WT gp4 samples, the percentage of LMW species is its central channel is 20% larger than the central channel of the significantly lower than for gp4⌬primase (supplemental Table heptamer. gp4⌬primase and WT gp4 display similar oligomer- S2). ization patterns at low protein concentration. ⌬ Supplemental Fig. S1B displays sw plotted as a function of the gp4 primase Binding to ssDNA—The binding of gp4 to a total protein concentration. In contrast to the data obtained for 27-mer oligonucleotide was measured in the presence of the ⌬ ␤ ␥ gp4 primase, the figure shows that sw increases with increasing nonhydrolysable , -methylene dTTP using a nitrocellulose concentration of WT gp4. Such concentration behavior is typ- DNA binding assay (Fig. 5A). WT gp4 binds the oligonucleotide Ϯ ⌬ ical for a chemically reacting system. In summary, the results with a Kd of 161 47 nM. In comparison, gp4 primase binds Ϯ suggest the primase facilitates oligomerization of DNA heli- the ssDNA 3-fold less tightly (Kd of 411 53 nM) and binds 30% case. The high percentages of LMW species detected for of the oligonucleotide at the highest protein concentration. The gp4⌬primase indicate that the primase stabilizes the ring of 3-fold poorer binding of gp4⌬primase to ssDNA is likely due to DNA helicase. the fact that gp4⌬primase does not contain the RPD that alone Analysis of gp4⌬primase Oligomerization by Electron Micro- can bind ssDNA (31). It was proposed earlier that the primase scopy—We used negative staining EM to examine the oligo- facilitates loading of gp4 onto ssDNA (32). merization properties of gp4⌬primase and WT gp4. gp4 As shown in the previous sections, gp4 exists in multiple samples were incubated in the buffer previously used for sedi- oligomeric forms, but the functional form is a hexamer. Only mentation velocity AUC. A representative image of the nega- hexamers are found to bind to ssDNA (30). Oligomerization of tively stained sample is shown in Fig. 4C. Image classification WT gp4 in the presence of ssDNA was examined earlier by revealed the presence of three different species (Fig. 4D). Hexa- PAGE under native conditions (17). Two bands seen in the gels meric and heptameric rings are present in both samples (34 and represent the hexameric and heptameric forms. In the presence 52%, respectively, for gp4⌬primase and 37 and 48%, respec- of ssDNA and ␤,␥-methylene dTTP, the slower migrating band tively, for WT gp4). A significant fraction of particles (14% for disappears, indicative of conversion from the heptameric to the gp4⌬primase and 15% for the WT gp4) display octameric rings. hexameric ring of gp4 (30). In the current study, we assessed the The octameric ring is 10% larger than the heptameric ring, and effect of ssDNA on the oligomerization of gp4⌬primase. Differ-

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⌬

FIGURE 5. Characterization of ssDNA binding, DNA unwinding, and strand displacement synthesis of gp4 primase. A, binding to ssDNA was measured Downloaded from in a nitrocellulose DNA binding assay. The reactions contained different concentrations of gp4⌬primase or WT gp4, 5Ј-32P-labeled 27-mer oligonucleotide, and ␤,␥-methylene dTTP. The quantity of protein bound ssDNA and free ssDNA was measured in a PhosphorImager. The graph shows percentages of bound ssDNA as a function of increasing concentrations of gp4⌬primase (filled circles) and WT gp4 (open circles). B, oligomerization of gp4⌬primase upon binding to ssDNA was assessed by native gel electrophoresis. Three different protein concentrations were incubated in the presence or absence of a 27-mer oligonucleotide in ␤ ␥ buffer containing MgCl2 and , -methylene dTTP and then cross-linked with glutaraldehyde. The products of the cross-linking reaction were analyzed on the native gel. C, DNA unwinding activity of gp4⌬primase was measured by the release of the 32P-labeled single-stranded (ss) oligonucleotide from the double- stranded (ds) DNA substrate shown in the inset and described in the text. The gel and graph show the amount of 37-mer oligonucleotide released from the DNA substrate in reaction mixtures containing increasing concentrations of gp4⌬primase (filled circles on the graph) or WT gp4 (open circles). D, strand displacement http://www.jbc.org/ activity of gp4⌬primase was measured in a minicircle assay. A minicircle DNA (inset) containing a replication fork was prepared as described previously and used as a primer/template (28). The reactions contained the minicircle DNA, the four dNTPs with [␣-32P]dGTP, gp5/trx, and gp4⌬primase or WT gp4. In the control reaction gp4 was omitted. Incorporation of [␣-32P]dGTP into the minicircle DNA is shown as the histogram.

ent protein concentrations were incubated in the presence or single-molecule assay (Fig. 3). In the single-molecule assay,

absence of a 27-mer oligonucleotide and ␤,␥-methylene dTTP. we can simultaneously monitor only several individual repli- by guest on October 5, 2019 The protein was then cross-linked with glutaraldehyde. In the somes. Consequently, the protein and DNA concentrations absence of ssDNA, we observe two protein bands on the native used are relatively low, making it difficult to investigate the gel (Fig. 5B). Upon the addition of ssDNA, the slower-migrating activity of proteins with low binding affinity to DNA. There- band disappears, representing the conversion of the heptameric fore, we have examined the ability of gp4⌬primase to medi- rings to hexamers. ate leading strand synthesis using a DNA minicircle assay DNA Unwinding Activity and Strand Displacement Synthesis (18). The minicircle, depicted in the inset of Fig. 5D, consists with gp5/trx—Once loaded onto ssDNA, gp4 translocates in a of a 70-bp circular dsDNA with a replication fork and a 5Ј 5Ј to 3Ј direction and unwinds dsDNA using energy obtained tail onto which gp4 can assemble. The incorporation of from dTTP hydrolysis. The dTTP hydrolysis activity of [32P]dGMP allows for the measurement of leading strand gp4⌬primase and WT gp4 are the same (27). In this section synthesis. Using this assay, we do detect DNA synthesis by we compare the unwinding activity of both proteins. the gp4⌬primase-gp5/trx complex (15% of that measured for The DNA substrate for the unwinding assay was prepared by WT gp4). annealing a 32P-labeled 37-mer oligonucleotide with a circular Small Angle X-ray Scattering Structure of the Leading Strand M13 DNA such that a 22-nt double-helical fragment is formed, Complex—We have used SAXS to analyze a solution structure as well as a 5Ј tail of 15 nucleotides (Fig. 5C, inset). Reaction of the complex between gp4⌬primase, gp5/trx, and a primer/ mixtures containing the DNA substrate described above, template. The SPR experiments presented in Fig. 2 show that dTTP, and different concentrations of gp4⌬primase or WT gp4 stable complexes of the two proteins form in the presence of a were incubated for 10 min and then stopped and analyzed on primer/template, ddATP and the next incoming nucleotide the gel (Fig. 5C, gel). Intensities of ssDNA and dsDNA bands (Fig. 2) (8). Therefore, samples for SAXS were prepared in an were measured in a PhosphorImager. At WT gp4 of 0.24 ␮M, analogous manner. 50% of the 37-mer is displaced from the DNA substrate. In gp5/trx bound to a primer/template in the presence of contrast, we estimate that 1 ␮M gp4⌬primase is required for ddATP and ddGTP was mixed with gp4⌬primase previously unwinding of the identical amount of DNA (Fig. 5C, graph). incubated with 15-mer single-stranded oligonucleotide in gp4⌬primase is five times less active than WT gp4 in unwinding buffer containing ␤,␥-methylene dTTP. The 15-mer was DNA. included to ensure a uniform oligomerization pattern of The low binding affinity of gp4⌬primase to ssDNA and the gp4⌬primase (Fig. 5B). The SAXS spectrum acquired for the resultant low unwinding activity account for the lack of complex described above and the corresponding Guinier plot detectable leading strand DNA synthesis measured in the are presented in Fig. 6 (A and B, respectively). Linearity of the

39056 JOURNAL OF BIOLOGICAL CHEMISTRY VOLUME 287•NUMBER 46•NOVEMBER 9, 2012 DNA Polymerase-DNA Helicase Interaction Downloaded from

FIGURE 6. Small angle x-ray scattering structure of the leading strand complex (gp5/trx-primer/template-gp4⌬primase-ssDNA). The sample for SAXS measurements was prepared by mixing gp4⌬primase bound to a 15-mer oligonucleotide in the presence of ␤,␥-methylene dTTP with gp5/trx bound to a primer/template in the presence of ddATP and dGTP. A, the scattering curve measured for the leading strand complex. B, the corresponding Guinier plot. C, distance distribution function p(r) of the leading strand complex has a Dmax value of 180 Å. D, the averaged ab initio model of the leading strand complex. Crystal structures of the hexameric DNA helicase (shown in blue; Protein Data Bank accession number 1CR0) and gp5/trx bound to a primer/template (polymerase and exonuclease domain of gp5 shown in red and pink, respectively; trx displayed in orange; primer/template shown in black; Protein Data Bank http://www.jbc.org/ accession number 1T7P) were docked into the SAXS envelope using the MultiFit method in the program Chimera. The model of the 17-amino acid C-terminal tail of gp4 that did not diffract in crystal was built in PyMol and is displayed as a yellow surface. Residues from the basic loading patch are colored in white. E, choreography of a replication fork modeled onto the leading strand complex. The residues from the subunit interface ␤-hairpin with Phe-523 are shown as the orange patch at the front surface of DNA helicase (see “Discussion”).

2 Ϫ2

very small angle part of the spectrum (q Ն 0.00064 Å ) indi- final averaged model, the first low resolution structure of the by guest on October 5, 2019 cates no aggregation. An upward deviation from linearity in the complex between gp5/trx, and DNA helicase, is shown in Fig. wider angle portion of the spectrum (Ͼ0.004 ÅϪ2) corresponds 6D. The crystal structures of the hexameric DNA helicase (33) to more pronounced aberrations from internal symmetry. The and gp5/trx bound to a primer/template (6) were docked into calculated hydrated radius of gyration (Rg) for the leading the SAXS envelope using the MultiFit method in the program strand complex is 60.5 Å (supplemental Table S3). We also Chimera (34). The best structural model obtained by the fitting carried out control SAXS measurements of samples containing procedure is presented in Fig. 6 (D and E). In the model, the tip gp5/trx-primer/template or gp4⌬primase-ssDNA. The calcu- of the palm domain of gp5 (red) docks into the cleft formed by

lated Rg values are 34 and 43 Å, respectively. The distance dis- the two surfaces in the neighboring subunits within the hexa- tribution function (p(r)) measured for the leading strand com- meric ring of DNA helicase (blue). Such orientation of gp5/trx plex is presented in Fig. 6C. To obtain a reliable result of the and the DNA helicase allows for binding of the C-terminal tail maximum diameter (Dmax), we used a search approach. In this of the helicase (yellow) to a basic patch on the front of the method, p(r) was calculated from SAXS data when Dmax was set polymerase (white). as a variable in the range of 100–300 Å with increments of 10 Å. DISCUSSION Evaluation parameters were analyzed, and the values of Dmax that showed the best overall behavior and GNOM statistics During leading strand synthesis, gp5/trx and gp4 make were chosen for further analysis (supplemental Fig. S2). The numerous interactions. gp5/trx joins the replisome by binding

p(r) of the leading strand complex shows a Dmax of 180 Å. The to the C-terminal acidic tail of gp4 via its basic loading patch bell-like shape of the p(r) indicates a compact sphere-like struc- (Fig. 7A). Alternatively, gp5/trx first binds the C-terminal tail ⌬ ture. The Dmax values for the samples containing gp4 through a basic exchange patch (Fig. 7B), but it must still switch primase-ssDNA and gp5/trx-primer/template are 126 and its binding to the loading patch to initiate DNA synthesis (11). 105 Å, respectively (supplemental Table S3). We also used Elimination of the basic charges in the exchange patch does not SAXS to investigate the interactions between gp5/trx bound affect strand displacement synthesis (9). Once leading strand to a primer/template with WT gp4, gp4⌬ZBD, or gp4⌬C. synthesis is initiated, a highly stable complex of gp5/trx and gp4 Attempts to obtain structures for the above complexes were is formed, increasing the processivity to ϳ5 kb/binding event unsuccessful because of aggregation of the proteins at high (10) (Fig. 7C). gp5/trx alone has a processivity of 800 nucleo- protein concentration. tides when copying ssDNA (12), and gp5 in the absence of trx We have used the software GASBOR to reconstruct ab initio has a processivity of less than 15 nucleotides (15). However, 10 structural models of the leading strand complex (24). The gp5/trx does occasionally dissociate from this stable complex

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FIGURE 7. The specific interactions of gp5/trx and gp4 coordinate each step in DNA replication. A, the gp4 assembles as a hexamer on the lagging strand Downloaded from DNA. gp5/trx is recruited to the replication fork by an initial interaction with the acidic C-terminal tail of gp4 through its loading patch containing basic residues. B, gp5/trx can also initially bind to the C-terminal tail of gp4 via its exchange patch located within the thioredoxin-binding domain (TBD) and containing two basic loops. C, the interaction of the loading patch of gp5 with the C-terminal tail of helicase facilitates the loading of gp5/trx to the primer/template, and the interaction is maintained during leading strand synthesis. gp5/trx and gp4 also interact through the helicase domain of gp4 and a part of the surface of the polymerase domain of gp5/trx. D, the C-terminal tails of the gp4 hexamer not only bind any dissociating gp5/trx, but they also accommodate additional gp5/trx through interactions with the exchange patch of gp5. Any of these gp5/trx can be reloaded onto the primer/template without involvement of the loading patch. http://www.jbc.org/

approximately every 5 kb. If gp5/trx does dissociate into solu- sedimentation velocity measurements support the above con- tion, it can still be replaced by a gp5/trx already bound by its clusions and show that removal of the primase domain affects exchange patch to a C-terminal tail of one of the subunits of the the dynamic equilibrium between various oligomeric forms of

hexameric gp4 (Fig. 7D). Thus the interaction of the C-terminal the helicase. A similar stabilization effect of the primase on the by guest on October 5, 2019 tail with the exchange patch of gp5 increases the processivity to helicase can be observed in the crystal structure of B. stearo- Ͼ17 kb (9). thermophilus primase/helicase complex (25). In the structure, Lacking to date has been an identification of the stable com- the three helicase binding domains of the primase stabilize a plex formed between gp5/trx and gp4 during leading strand conformation of the N-terminal domain of the helicase. synthesis. This complex that mediates leading strand synthesis Additionally, the primase is important for gp4 binding to is extremely stable with a half-life of more than 10 min (8). The ssDNA and for dsDNA unwinding. gp4⌬primase binds the binding between gp4 and ssDNA is stable, with a dissociation ssDNA ϳ3-fold less tightly and displays five times less unwind- rate of 0.002 sϪ1 (35). However, the complex of gp5/trx, in the ing activity than WT gp4. The reduction in DNA binding and absence of gp4, readily dissociates from a primer/template with unwinding activities is due to the removal of the RPD from gp4. a dissociation constant of 0.2 sϪ1 (36). Consequently, there is an We showed previously that gp4⌬ZBD retains full helicase activ- interaction of gp5/trx with gp4 that accounts for the association ity and displays ssDNA binding comparable with the WT pro- observed during leading strand synthesis. tein (16). It was proposed earlier that the primase facilitates In the current study we show that the stable complex is medi- loading of gp4 onto ssDNA (32). The five-step model involves ated through the interaction of the helicase to the palm domain the fast initial binding of the primase to the primase recognition of gp5 and an electrostatic binding of the C-terminal tail of the sequence in DNA followed by a rapid conformational change in helicase to the loading patch of gp5. The ZBD and RPD are not gp4 that triggers ring opening, movement of the DNA into the directly involved in binding as indicated by nearly identical central channel of the helicase ring, and ring closure. The cur- ⌬ Rmax values measured using SPR for WT gp4, gp4 ZBD, and rent study is consistent with this model. gp4⌬primase, as well as the lack of binding measured with We propose that an electrostatic interaction of the C-termi- gp4⌬helicase. The primase domain of gp4 is involved in forma- nal tail of the helicase to the loading patch located on the front tion of the leading strand complex by stabilizing the helicase. of the polymerase is maintained during leading strand synthe- gp4⌬primase associates with gp5/trx-primer/template in a sis. Removal of the C-terminal tail of gp4 decreases the binding similar way as WT gp4; however, its dissociation from the com- of gp4 to the leading strand complex. In addition, elimination of plex is faster. Removal of the ZBD from gp4 has a minimal the interaction described above abolishes initiation of strand influence on leading strand synthesis as measured in the single- displacement synthesis and reduces the rate of ongoing synthe- molecule assay. Removal of both the ZBD and the RPD results sis by 50% (11). We estimate that the interaction between the in a significant reduction of leading strand synthesis indicative C-terminal tail of the helicase and a basic patch on gp5 accounts of the major role of the RPD in stabilization of the helicase. The for 30% of the binding between gp4 and the leading strand com-

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plex as evidenced by SPR measurements with gp4⌬C. The that catalyzes homologous pairing during meiosis. Structural remaining 70% of binding is due to the interaction of the heli- analysis of Dms1 revealed that this protein forms both hepta- case and the palm domain of gp5. We measured using SPR that meric and octameric rings (43, 44), as well as helical filaments gp4⌬C⌬primase binds to the leading strand complex at 30% of with a different number of monomers per helical turn depen- the level observed with WT gp4. Therefore, we estimate that ding on the DNA template to which it is bound (45). What is the the presence of the primase stabilizes the helicase, contributing functional relevance of the octameric gp4? Is it enzymatically 40% of the binding between gp4 and gp5/trx during leading active, or perhaps various oligomerization patterns are simply a strand synthesis. natural consequence of flexible subunit interfaces? Structural We present the first low resolution structure of the complex similarities between gp4 and the recombination proteins bring between DNA polymerase and DNA helicase solved using an intriguing possibility of the involvement of gp4 in DNA SAXS. In the SAXS model, the protein-protein interface recombination and repair. includes the gp5 surface located at the palm domain in the close In conclusion, we have identified the interaction between the proximity to helices: R, Q, and G1 and the cleft between the two DNA polymerase and DNA helicase that is essential for the high neighboring gp4 subunits of the hexameric ring (Fig. 6E). The processivity of leading strand synthesis, and described it in the relative orientation of gp5/trx and the DNA helicase in the context of other specific interactions that govern the interplay complex allows for binding of the C-terminal tail of the helicase between these two enzymes. We hope that understanding of to a basic patch on the front of the polymerase. In the SAXS the replication process in bacteriophage T7 will facilitate stud- model a primer/template bound to gp5/trx (displayed in black ies in more complex systems. in Fig. 6E) is positioned such that the extension of the 5Ј-end of the ssDNA template would position it in the close proximity to Acknowledgment—We thank Steven Moskowitz for assistance in Downloaded from the subunit interface ␤-hairpin that contains residue Phe-523 preparation of figures. (orange patch at the front surface of DNA helicase). We dem- onstrated earlier that the subunit interface ␤-hairpin, specifi- cally Phe-523, interacts directly with the extruded DNA leading REFERENCES strand during the process of DNA unwinding (17). The lagging 1. Hamdan, S. M., and Richardson, C. C. (2009) Motors, switches, and con- http://www.jbc.org/ strand passes through the central channel in the hexameric ring tacts in the replisome. Annu. Rev. Biochem. 78, 205–243 2. Bernstein, J. A., and Richardson, C. C. (1988) A 7-kDa region of the bac- of DNA helicase, making interactions with the residues in the ␤ teriophage T7 gene 4 protein is required for primase but not for helicase central -hairpins located at the inner surface of the channel. activity. Proc. Natl. Acad. Sci. U.S.A. 85, 396–400 At the replication fork, a 5Ј to 3Ј forward motion of gp4 relative 3. Matson, S. W., Tabor, S., and Richardson, C. C. (1983) The gene 4 protein

to the lagging strand to which it is bound unwinds the duplex of bacteriophage T7. Characterization of helicase activity. J. Biol. Chem. by guest on October 5, 2019 DNA that it encounters. In the model, the extruded DNA 258, 14017–14024 strand has the correct polarity and position to bind at the DNA- 4. Romano, L. J., and Richardson, C. C. (1979) Characterization of the ribo- Ј nucleic acid primers and the deoxyribonucleic acid product synthesized binding crevice of gp5 such that the 3 -terminus of the leading by the DNA polymerase and gene 4 protein of bacteriophage T7. J. Biol. strand primer is situated in the active site. This configuration of Chem. 254, 10483–10489 the replication fork is stabilized by the two interactions 5. Huber, H. E., Tabor, S., and Richardson, C. C. (1987) Escherichia coli between gp5/trx and DNA helicase described in the present thioredoxin stabilizes complexes of bacteriophage T7 DNA polymerase study. and primed templates. J. Biol. Chem. 262, 16224–16232 gp4 has been crystallized in three different oligomeric forms. 6. Doublie´, S., Tabor, S., Long, A. M., Richardson, C. C., and Ellenberger, T. ⌬ (1998) Crystal structure of a bacteriophage T7 DNA replication complex The structure of gp4 ZBD revealed a heptameric arrangement at 2.2 A resolution. Nature 391, 251–258 of the ring subunits (29). In contrast, gp4⌬primase was crystal- 7. Akabayov, B., Akabayov, S. R., Lee, S. J., Tabor, S., Kulczyk, A. W., and lized as a hexamer (33). Deletion of an additional 31 amino Richardson, C. C. (2010) Conformational dynamics of bacteriophage T7 acids from the N terminus of the gp4⌬primase results in the DNA polymerase and its processivity factor, Escherichia coli thioredoxin. formation of a helical filament with six monomers per helical Proc. Natl. Acad. Sci. U.S.A. 107, 15033–15038 8. Hamdan, S. M., Marintcheva, B., Cook, T., Lee, S. J., Tabor, S., and Rich- turn (37). Here, we have identified yet another oligomeric form ⌬ ardson, C. C. (2005) A unique loop in T7 DNA polymerase mediates the of gp4. The EM analysis of WT gp4 and gp4 primase revealed binding of helicase-primase, DNA binding protein, and processivity fac- the presence of octameric rings in the samples. Similar struc- tor. Proc. Natl. Acad. Sci. U.S.A. 102, 5096–5101 tural diversity has been observed with proteins involved in 9. Hamdan, S. M., Johnson, D. E., Tanner, N. A., Lee, J. B., Qimron, U., Tabor, DNA recombination. In bacterial homologous recombination, S., van Oijen, A. M., and Richardson, C. C. (2007) Dynamic DNA helicase- RecA protein is a key enzyme that mediates a homology search DNA polymerase interactions assure processive replication fork movement. Mol. Cell 27, 539–549 and exchange between two DNA molecules. RecA protein 10. Loparo, J. J., Kulczyk, A. W., Richardson, C. C., and van Oijen, A. M. (2011) exists in two different oligomeric forms. In the crystal E. coli Simultaneous single-molecule measurements of phage T7 replisome RecA forms helical filaments (38), but the EM studies of RecA composition and function reveal the mechanism of polymerase exchange. under similar conditions revealed the presence of hexameric Proc. Natl. Acad. Sci. U.S.A. 108, 3584–3589 rings (39). The archaeal homolog of RecA, Sulfolobus solfatari- 11. Zhang, H., Lee, S. J., Zhu, B., Tran, N. Q., Tabor, S., and Richardson, C. C. cus RadA protein, can form octameric rings or helical filaments (2011) Helicase-DNA polymerase interaction is critical to initiate leading- strand DNA synthesis. Proc. Natl. Acad. Sci. U.S.A. 108, 9372–9377 (40). The Pyrococcus furiosus RadA protein (PfRad51) was crys- 12. Lee, J. B., Hite, R. K., Hamdan, S. M., Xie, X. S., Richardson, C. C., and van tallized as a heptamer (41), and EM studies revealed hexameric Oijen, A. M. (2006) DNA primase acts as a molecular brake in DNA filaments (42). The human Dmc1 protein is a RecA homolog replication. Nature 439, 621–624

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13. Notarnicola, S. M., and Richardson, C. C. (1993) The nucleotide binding crystal structure of the bifunctional primase-helicase of bacteriophage T7. site of the helicase/primase of bacteriophage T7. Interaction of mutant Mol. Cell 12, 1113–1123 and wild-type proteins. J. Biol. Chem. 268, 27198–27207 30. Crampton, D. J., Ohi, M., Qimron, U., Walz, T., and Richardson, C. C. 14. Notarnicola, S. M., Mulcahy, H. L., Lee, J., and Richardson, C. C. (1997) The (2006) Oligomeric states of bacteriophage T7 gene 4 primase/helicase. J. acidic carboxyl terminus of the bacteriophage T7 gene 4 helicase/primase Mol. Biol. 360, 667–677 interacts with T7 DNA polymerase. J. Biol. Chem. 272, 18425–18433 31. Kato, M., Ito, T., Wagner, G., and Ellenberger, T. (2004) A molecular 15. Tabor, S., Huber, H. E., and Richardson, C. C. (1987) Escherichia coli handoff between bacteriophage T7 DNA primase and T7 DNA polymer- thioredoxin confers processivity on the DNA polymerase activity of the ase initiates DNA synthesis. J. Biol. Chem. 279, 30554–30562 gene 5 protein of bacteriophage T7. J. Biol. Chem. 262, 16212–16223 32. Ahnert, P., Picha, K. M., and Patel, S. S. (2000) A ring-opening mechanism 16. Kulczyk, A. W., and Richardson, C. C. (2012) Molecular interactions in the for DNA binding in the central channel of the T7 helicase-primase pro- priming complex of bacteriophage T7. Proc. Natl. Acad. Sci. U.S.A. 109, tein. EMBO J. 19, 3418–3427 9408–9413 33. Singleton, M. R., Sawaya, M. R., Ellenberger, T., and Wigley, D. B. (2000) 17. Satapathy, A. K., Kulczyk, A. W., Ghosh, S., van Oijen, A. M.,and Richard- Crystal structure of T7 gene 4 ring helicase indicates a mechanism for son, C. C. (2011) A critical residue for coupling dTTP hydrolysis with sequential hydrolysis of nucleotides. Cell 101, 589–600 DNA unwinding by the helicase of bacteriophage T7. J. Biol. Chem. 286, 34. Lasker, K., Sali, A., and Wolfson, H. J. (2010) Determining macromolecu- 34468–34478 lar assembly structures by molecular docking and fitting into an electron 18. Lee, J., Chastain, P. D., 2nd, Kusakabe, T., Griffith, J. D., and Richardson, density map. Proteins 78, 3205–3211 C. C. (1998) Coordinated leading and lagging strand DNA synthesis on a 35. Kim, D. E., Narayan, M., and Patel, S. S. (2002) T7 DNA helicase. A mo- minicircular template. Mol. Cell 1, 1001–1010 lecular motor that processively and unidirectionally translocates along 19. Schuck, P. (2000) Size-distribution analysis of macromolecules by sedi- single-stranded DNA. J. Mol. Biol. 321, 807–819 mentation velocity ultracentrifugation and lamm equation modeling. Bio- 36. Patel, S. S., Wong, I., and Johnson, K. A. (1991) Pre-steady-state kinetic phys. J. 78, 1606–1619 analysis of processive DNA replication including complete characteriza- 20. Ohi, M., Li, Y., Cheng, Y., and Walz, T. (2004) Negative staining and image Downloaded from tion of an exonuclease-deficient mutant. Biochemistry 30, 511–525 classification. Powerful tools in modern electron microscopy. Biol. Proced. 37. Sawaya, M. R., Guo, S., Tabor, S., Richardson, C. C., and Ellenberger, T. Online 6, 23–34 (1999) Crystal structure of the helicase domain from the replicative heli- 21. Chen, J. Z., and Grigorieff, N. (2007) SIGNATURE. A single-particle se- case-primase of bacteriophage T7. Cell 99, 167–177 lection system for molecular electron microscopy. J. Struct. Biol. 157, 38. Story, R. M., Weber, I. T., and Steitz, T. A. (1992) The structure of the 168–173 E. coli recA protein monomer and polymer. Nature 355, 318–325 22. Hohn, M., Tang, G., Goodyear, G., Baldwin, P. R., Huang, Z., Penczek, http://www.jbc.org/ 39. Yu, X., and Egelman, E. H. (1997) The RecA hexamer is a structural ho- P. A., Yang, C., Glaeser, R. M., Adams, P. D., and Ludtke, S. J. (2007) mologue of ring helicases. Nat. Struct. Biol. 4, 101–104 SPARX, a new environment for Cryo-EM image processing. J. Struct. Biol. 40. Yang, S., Yu, X., Seitz, E. M., Kowalczykowski, S. C., and Egelman, E. H. 157, 47–55 23. Lipfert, J., Das, R., Chu, V. B., Kudaravalli, M., Boyd, N., Herschlag, D., and (2001) Archaeal RadA protein binds DNA as both helical filaments and Doniach, S. (2007) Structural transitions and thermodynamics of a gly- octameric rings. J. Mol. Biol. 314, 1077–1085 cine-dependent riboswitch from Vibrio cholerae. J. Mol. Biol. 365, 41. Shin, D. S., Pellegrini, L., Daniels, D. S., Yelent, B., Craig, L., Bates, D., Yu, by guest on October 5, 2019 1393–1406 D. S., Shivji, M. K., Hitomi, C., Arvai, A. S., Volkmann, N., Tsuruta, H., 24. Svergun, D. I., Petoukhov, M. V., and Koch, M. H. (2001) Determination of Blundell, T. L., Venkitaraman, A. R., and Tainer, J. A. (2003) Full-length domain structure of proteins from X-ray solution scattering. Biophys. J. archaeal Rad51 structure and mutants. Mechanisms for RAD51 assembly 80, 2946–2953 and control by BRCA2. EMBO J. 22, 4566–4576 25. Bailey, S., Eliason, W. K., and Steitz, T. A. (2007) Structure of hexameric 42. Komori, K., Miyata, T., DiRuggiero, J., Holley-Shanks, R., Hayashi, I., DnaB helicase and its complex with a domain of DnaG primase. Science Cann, I. K., Mayanagi, K., Shinagawa, H., and Ishino, Y. (2000) Both RadA 318, 459–463 and RadB are involved in homologous recombination in Pyrococcus furio- 26. Kulczyk, A. W., Tanner, N. A., Loparo, J. J., Richardson, C. C., and van sus. J. Biol. Chem. 275, 33782–33790 Oijen, A. M. (2010) Direct observation of enzymes replicating DNA using 43. Kinebuchi, T., Kagawa, W., Enomoto, R., Tanaka, K., Miyagawa, K., Shi- a single-molecule DNA stretching assay. J. Vis. Exp. 23, 37 bata, T., Kurumizaka, H., and Yokoyama, S. (2004) Structural basis for 27. Johnson, D. E., Takahashi, M., Hamdan, S. M., Lee, S. J., and Richardson, octameric ring formation and DNA interaction of the human homolo- C. C. (2007) Exchange of DNA polymerases at the replication fork of gous-pairing protein Dmc1. Mol. Cell 14, 363–374 bacteriophage T7. Proc. Natl. Acad. Sci. U.S.A. 104, 5312–5317 44. Passy, S. I., Yu, X., Li, Z., Radding, C. M., Masson, J. Y., West, S. C., and 28. Bird, L. E., Pan, H., Soultanas, P., and Wigley, D. B. (2000) Mapping pro- Egelman, E. H. (1999) Human Dmc1 protein binds DNA as an octameric tein-protein interactions within a stable complex of DNA primase and ring. Proc. Natl. Acad. Sci. U.S.A. 96, 10684–10688 DnaB helicase from Bacillus stearothermophilus. Biochemistry 39, 45. Okorokov, A. L., Chaban, Y. L., Bugreev, D. V., Hodgkinson, J., Mazin, 171–182 A. V., and Orlova, E. V. (2010) Structure of the hDmc1-ssDNA filament 29. Toth, E. A., Li, Y., Sawaya, M. R., Cheng, Y., and Ellenberger, T. (2003) The reveals the principles of its architecture. PLoS One 5, e8586

39060 JOURNAL OF BIOLOGICAL CHEMISTRY VOLUME 287•NUMBER 46•NOVEMBER 9, 2012 An Interaction between DNA Polymerase and Helicase Is Essential for the High Processivity of the Bacteriophage T7 Replisome Arkadiusz W. Kulczyk, Barak Akabayov, Seung-Joo Lee, Mihnea Bostina, Steven A. Berkowitz and Charles C. Richardson J. Biol. Chem. 2012, 287:39050-39060. doi: 10.1074/jbc.M112.410647 originally published online September 12, 2012

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