RNA Primer Handoff in Bacteriophage T4 DNA Replication
2008; Elsevier BV; Volume: 283; Issue: 33 Linguagem: Inglês
10.1074/jbc.m802762200
ISSN1083-351X
AutoresScott W. Nelson, Ravindra Kumar, Stephen J. Benkovic,
Tópico(s)Protein Structure and Dynamics
ResumoIn T4 phage, coordinated leading and lagging strand DNA synthesis is carried out by an eight-protein complex termed the replisome. The control of lagging strand DNA synthesis depends on a highly dynamic replisome with several proteins entering and leaving during DNA replication. Here we examine the role of single-stranded binding protein (gp32) in the repetitive cycles of lagging strand synthesis. Removal of the protein-interacting domain of gp32 results in a reduction in the number of primers synthesized and in the efficiency of primer transfer to the polymerase. We find that the primase protein is moderately processive, and this processivity depends on the presence of full-length gp32 at the replication fork. Surprisingly, we find that an increase in the efficiency of primer transfer to the clamp protein correlates with a decrease in the dissociation rate of the primase from the replisome. These findings result in a revised model of lagging strand DNA synthesis where the primase remains as part of the replisome after each successful cycle of Okazaki fragment synthesis. A delay in primer transfer results in an increased probability of the primase dissociating from the replication fork. The interplay between gp32, primase, clamp, and clamp loader dictates the rate and efficiency of primer synthesis, polymerase recycling, and primer transfer to the polymerase. In T4 phage, coordinated leading and lagging strand DNA synthesis is carried out by an eight-protein complex termed the replisome. The control of lagging strand DNA synthesis depends on a highly dynamic replisome with several proteins entering and leaving during DNA replication. Here we examine the role of single-stranded binding protein (gp32) in the repetitive cycles of lagging strand synthesis. Removal of the protein-interacting domain of gp32 results in a reduction in the number of primers synthesized and in the efficiency of primer transfer to the polymerase. We find that the primase protein is moderately processive, and this processivity depends on the presence of full-length gp32 at the replication fork. Surprisingly, we find that an increase in the efficiency of primer transfer to the clamp protein correlates with a decrease in the dissociation rate of the primase from the replisome. These findings result in a revised model of lagging strand DNA synthesis where the primase remains as part of the replisome after each successful cycle of Okazaki fragment synthesis. A delay in primer transfer results in an increased probability of the primase dissociating from the replication fork. The interplay between gp32, primase, clamp, and clamp loader dictates the rate and efficiency of primer synthesis, polymerase recycling, and primer transfer to the polymerase. The T4 4The abbreviations used are:T4bacteriophage T4gp32T4 single-stranded DNA-binding proteinssDNAsingle-stranded DNAdsDNAdouble-stranded DNAwt-gp32wild-type gp32gp32-Aresidues 1-253 of gp32gp32-Bresidues 22-301. replisome has served as a highly useful model system for studying coupled DNA replication (1Nossal N.G. FASEB J. 1992; 6: 871-878Crossref PubMed Scopus (86) Google Scholar). The T4 replisome is made up of eight proteins, all of which have counterparts in more complex organisms such as Escherichia coli, Saccharomyces cerevisiae, and humans (2Benkovic S.J. Valentine A.M. Salinas F. Annu. Rev. Biochem. 2001; 70: 181-208Crossref PubMed Scopus (282) Google Scholar). DNA synthesis is carried out in a 5′ to 3′ direction by T4 DNA polymerase (gp43), which together with the clamp protein (gp45) makes up the holoenzyme complex (3Kaboord B.F. Benkovic S.J. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 10881-10885Crossref PubMed Scopus (42) Google Scholar). The holoenzyme can form through several different routes, all dependent on the activity of the clamp loader protein (gp44/62) (4Smiley R.D. Zhuang Z. Benkovic S.J. Hammes G.G. Biochemistry. 2006; 45: 7990-7997Crossref PubMed Scopus (37) Google Scholar, 5Zhuang Z. Berdis A.J. Benkovic S.J. Biochemistry. 2006; 45: 7976-7989Crossref PubMed Scopus (23) Google Scholar). The clamp loader is an AAA+ protein that uses energy derived from ATP hydrolysis to chaperone the holoenzyme assembly process (6Kaboord B.F. Benkovic S.J. Biochemistry. 1996; 35: 1084-1092Crossref PubMed Scopus (49) Google Scholar). The T4 primosome moves along the lagging strand DNA template in the 5′ to 3′ direction and is composed of a hexameric helicase (gp41) that unwinds the duplex DNA and an oligomeric primase (gp61) that synthesizes pentaribonucleotide primers at 5′-GTT and 5′-GCT sequences to initiate repetitive Okazaki fragment synthesis (7Zhang Z. Spiering M.M. Trakselis M.A. Ishmael F.T. Xi J. Benkovic S.J. Hammes G.G. Proc. Natl. Acad. Sci. U. S. A. 2005; 102: 3254-3259Crossref PubMed Scopus (40) Google Scholar, 8Hinton D.M. Nossal N.G. J. Biol. Chem. 1987; 262: 10873-10878Abstract Full Text PDF PubMed Google Scholar). The helicase is loaded onto the lagging strand template by the helicase loader protein (gp59) (9Barry J. Alberts B. J. Biol. Chem. 1994; 269: 33049-33062Abstract Full Text PDF PubMed Google Scholar, 10Nossal N.G. Hinton D.M. Hobbs L.J. Spacciapoli P. Methods Enzymol. 1995; 262: 560-584Crossref PubMed Scopus (33) Google Scholar). gp59 plays an additional role as a "gatekeeper" of the replisome by coordinating the assembly of the primosome with the initiation of leading strand DNA synthesis through a direct interaction with the leading strand polymerase (11Dudas K.C. Kreuzer K.N. J. Biol. Chem. 2005; 280: 21561-21569Abstract Full Text Full Text PDF PubMed Scopus (24) Google Scholar, 12Xi J. Zhang Z. Zhuang Z. Yang J. Spiering M.M. Hammes G.G. Benkovic S.J. Biochemistry. 2005; 44: 7747-7756Crossref PubMed Scopus (37) Google Scholar, 13Nelson S.W. Yang J. Benkovic S.J. J. Biol. Chem. 2006; 281: 8697-8706Abstract Full Text Full Text PDF PubMed Scopus (14) Google Scholar). Finally, gp32 plays a central role in most aspects of DNA metabolism, including DNA replication (14Alberts B.M. Barry J. Bedinger P. Formosa T. Jongeneel C.V. Kreuzer K.N. Cold Spring Harbor Symp. Quant. Biol. 1983; 47: 655-668Crossref PubMed Google Scholar). The gp32 protein coats the ssDNA produced by the primosome and is thought to be involved in the coordination of lagging strand synthesis (15Salinas F. Benkovic S.J. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 7196-7201Crossref PubMed Scopus (54) Google Scholar). gp32 is made up of N-terminal, C-terminal, and core domains (16Waidner L.A. Flynn E.K. Wu M. Li X. Karpel R.L. J. Biol. Chem. 2001; 276: 2509-2516Abstract Full Text Full Text PDF PubMed Scopus (23) Google Scholar). The N-terminal domain (domain B for "basic") is involved in cooperative ssDNA binding. Removal of residues 1-21 completely eliminates ssDNA binding cooperativity (17Giedroc D.P. Khan R. Barnhart K. J. Biol. Chem. 1990; 265: 11444-11455Abstract Full Text PDF PubMed Google Scholar, 18Villemain J.L. Ma Y. Giedroc D.P. Morrical S.W. J. Biol. Chem. 2000; 275: 31496-31504Abstract Full Text Full Text PDF PubMed Scopus (10) Google Scholar). The major function of the highly acidic C-terminal domain (residues 254-301, domain A for "acidic") is to interact with other T4 proteins (19Krassa K.B. Green L.S. Gold L. Proc. Natl. Acad. Sci. U. S. A. 1991; 88: 4010-4014Crossref PubMed Scopus (42) Google Scholar). Affinity chromatography using gp32-agarose has detected interactions between gp32 and itself, gp43, gp45, and gp59 (14Alberts B.M. Barry J. Bedinger P. Formosa T. Jongeneel C.V. Kreuzer K.N. Cold Spring Harbor Symp. Quant. Biol. 1983; 47: 655-668Crossref PubMed Google Scholar, 20Morrical S.W. Beernink H.T. Dash A. Hempstead K. J. Biol. Chem. 1996; 271: 20198-20207Abstract Full Text Full Text PDF PubMed Scopus (44) Google Scholar). gp32 also has been shown to co-purify with gp61 (21Burke R.L. Munn M. Barry J. Alberts B.M. J. Biol. Chem. 1985; 260: 1711-1722Abstract Full Text PDF PubMed Google Scholar). Removal of domain A abolishes interaction with all of these proteins (19Krassa K.B. Green L.S. Gold L. Proc. Natl. Acad. Sci. U. S. A. 1991; 88: 4010-4014Crossref PubMed Scopus (42) Google Scholar). The core domain is responsible for the recognition and binding of ssDNA (22Shamoo Y. Friedman A.M. Parsons M.R. Konigsberg W.H. Steitz T.A. Nature. 1995; 376: 362-366Crossref PubMed Scopus (224) Google Scholar). bacteriophage T4 T4 single-stranded DNA-binding protein single-stranded DNA double-stranded DNA wild-type gp32 residues 1-253 of gp32 residues 22-301. The T4 replisome appears to be the most dynamic of the well characterized replisomes, with the primase, clamp loader, clamp, and ssDNA-binding protein all exchanging with solution proteins during coupled leading and lagging strand synthesis (23Kadyrov F.A. Drake J.W. J. Biol. Chem. 2001; 276: 29559-29566Abstract Full Text Full Text PDF PubMed Scopus (34) Google Scholar, 24Trakselis M.A. Roccasecca R.M. Yang J. Valentine A.M. Benkovic S.J. J. Biol. Chem. 2003; 278: 49839-49849Abstract Full Text Full Text PDF PubMed Scopus (26) Google Scholar). Additionally, the DNA polymerase is "dynamically processive," meaning solution polymerase is capable of displacing the replicating polymerase during active replication without prior disassembly of the holoenzyme complex (25Yang J. Zhuang Z. Roccasecca R.M. Trakselis M.A. Benkovic S.J. Proc. Natl. Acad. Sci. U. S. A. 2004; 101: 8289-8294Crossref PubMed Scopus (106) Google Scholar). Under normal conditions, only the hexameric helicase remains at the replication fork for the lifetime of the replisome (26Schrock R.D. Alberts B. J. Biol. Chem. 1996; 271: 16678-16682Abstract Full Text Full Text PDF PubMed Scopus (31) Google Scholar). It is thought that many of these dynamic processes are related to the mechanism of repeated lagging strand synthesis. However, very little is known about the timing and rates of protein dissociation or how they may control lagging strand synthesis. Because of the opposite polarities of the leading and lagging strand template, the two polymerase holoenzymes must copy their templates in opposite directions (27Alberts B.M. Philos. Trans. R Soc. Lond. B Biol. Sci. 1987; 317: 395-420Crossref PubMed Scopus (74) Google Scholar). This fact, coupled with the observation that the lagging strand polymerase is resistant to dilution, led to the proposal of the "trombone model" for DNA replication (14Alberts B.M. Barry J. Bedinger P. Formosa T. Jongeneel C.V. Kreuzer K.N. Cold Spring Harbor Symp. Quant. Biol. 1983; 47: 655-668Crossref PubMed Google Scholar). This model links the two holoenzyme complexes and loops the lagging strand template into a structure resembling a trombone slide so that replication on both strands can occur in the same apparent direction. This model adequately explains why the lagging strand is synthesized in short 1-2-kb Okazaki fragments, whereas the leading strand is continuous. The lagging strand polymerase must repeatedly release from its position on the lagging strand template and recycle to the newly synthesized primer to begin a new round of Okazaki fragment synthesis. Presumably, interactions between the lagging strand polymerase and other components at the replication fork allow the polymerase to remain as part of the replisome during the recycling process. The signal for lagging strand polymerase release and recycling has been a subject of intense investigation (28Yang J. Nelson S.W. Benkovic S.J. Mol. Cell. 2006; 21: 153-164Abstract Full Text Full Text PDF PubMed Scopus (50) Google Scholar, 29Carver T.E. Sexton D.J. Benkovic S.J. Biochemistry. 1997; 36: 14409-14417Crossref PubMed Scopus (25) Google Scholar, 30Chastain P.D. Makhov A.M. Nossal N.G. Griffith J.D. Mol. Cell. 2000; 6: 803-814Abstract Full Text Full Text PDF PubMed Scopus (29) Google Scholar, 31Hacker K.J. Alberts B.M. J. Biol. Chem. 1994; 269: 24221-24228Abstract Full Text PDF PubMed Google Scholar). Several models have been proposed to act as the trigger for lagging strand release and recycling. The two with the most support are the collision and the signaling models. The collision model states that the collision of the lagging strand polymerase with the 5′ end of the previous Okazaki fragment causes the primase to synthesize an RNA primer and the polymerase to release from the lagging strand template and recycle to the newly made primer. In the signaling model, the lagging strand polymerase releases from the DNA as the result of events that are associated with the synthesis or capture of the RNA primer. There is substantial support for both of these models, and it is likely that both mechanisms are operable during lagging strand synthesis (28Yang J. Nelson S.W. Benkovic S.J. Mol. Cell. 2006; 21: 153-164Abstract Full Text Full Text PDF PubMed Scopus (50) Google Scholar). Support for the collision model comes from data indicating that the dissociation rate of the holoenzyme is drastically increased upon collision with the 5′ end of both DNA and RNA primers (29Carver T.E. Sexton D.J. Benkovic S.J. Biochemistry. 1997; 36: 14409-14417Crossref PubMed Scopus (25) Google Scholar, 31Hacker K.J. Alberts B.M. J. Biol. Chem. 1994; 269: 24221-24228Abstract Full Text PDF PubMed Google Scholar). Recent work from our laboratory has demonstrated that new Okazaki fragments can be initiated without completion of the previous fragment, indicating that the polymerase recycling via collision is not necessary (28Yang J. Nelson S.W. Benkovic S.J. Mol. Cell. 2006; 21: 153-164Abstract Full Text Full Text PDF PubMed Scopus (50) Google Scholar). The responsiveness of primer utilization efficiency and Okazaki fragment size to the concentration of clamp and clamp loader led us to suggest that clamp loading onto the RNA primer could serve as the signal for lagging strand polymerase release and recycling (28Yang J. Nelson S.W. Benkovic S.J. Mol. Cell. 2006; 21: 153-164Abstract Full Text Full Text PDF PubMed Scopus (50) Google Scholar). Computer simulations of lagging strand synthesis using a simple stochastic model incorporating the rate of primase association, primer synthesis, and clamp loading onto the newly synthesized primer accurately described the observed distribution of Okazaki fragments produced in vitro (28Yang J. Nelson S.W. Benkovic S.J. Mol. Cell. 2006; 21: 153-164Abstract Full Text Full Text PDF PubMed Scopus (50) Google Scholar). If clamp loading does serve as the signal for lagging strand polymerase dissociation, then primer handoff must follow an indirect pathway where the primase transfers the primer to the clamp loader and clamp proteins before the release and recycling of the lagging strand polymerase. This pathway is similar to that as described in the E. coli system and contrasts that of the T7 replisome (32Yuzhakov A. Kelman Z. O'Donnell M. Cell. 1999; 96: 153-163Abstract Full Text Full Text PDF PubMed Scopus (194) Google Scholar, 33Kato M. Ito T. Wagner G. Richardson C.C. Ellenberger T. Mol. Cell. 2003; 11: 1349-1360Abstract Full Text Full Text PDF PubMed Scopus (93) Google Scholar). Here we report on experiments that highlight the interplay between the ssDNA-binding protein, primase, clamp, and clamp loader during the initiation of lagging strand synthesis. We examined the effect of removing the protein interaction domain of gp32 (gp32-A) on primer synthesis, primer utilization, and primase processivity. We found that total primer synthesis is drastically reduced in the presence of gp32-A, and a reduction in the efficiency of primer transfer from the primase to the polymerase is also observed. These effects combine to produce abnormally long Okazaki fragments with a broad distribution ranging from 0.2 to 10 kb. Using an inactive primase trap protein, we find that the rate of dissociation of the primase from the replisome is dependent on the concentration of clamp and clamp loader proteins, as well as the presence of intact gp32. The fast dissociation rate of primase in the presence of gp32-A can be compensated for by high concentrations of clamp and clamp loader. Based on these results, we present a more elaborate model for lagging strand DNA synthesis where the primase remains as part of the replisome through successful cycles Okazaki fragment initiation. If primer handoff is delayed, the primase has a higher probability of dissociating from the replication fork, and if this occurs, a new primase must enter the replisome from solution to begin a new round of primer synthesis. [α-32P]dCTP, [α-32P]dGTP, and [α-32P]CTP were purchased from PerkinElmer Life Sciences. Unlabeled ribonucleotides were purchased from Roche Applied Science. Bacteriophage T4 replication proteins gp41, gp61, gp43, gp44/62, gp45, gp32, and primase trap protein were purified as described previously (24Trakselis M.A. Roccasecca R.M. Yang J. Valentine A.M. Benkovic S.J. J. Biol. Chem. 2003; 278: 49839-49849Abstract Full Text Full Text PDF PubMed Scopus (26) Google Scholar, 34Yang J. Trakselis M.A. Roccasecca R.M. Benkovic S.J. J. Biol. Chem. 2003; 278: 49828-49838Abstract Full Text Full Text PDF PubMed Scopus (28) Google Scholar). The gp32-A-petIMPACT and gp32-B-petIMPACT plasmids were provided by Jingsong Yang. The minicircle substrate was prepared as described previously (15Salinas F. Benkovic S.J. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 7196-7201Crossref PubMed Scopus (54) Google Scholar). Single-stranded M13 phage DNA (ssM13) was purified from infected XL1-Blue cells by polyethylene glycol precipitation and phenol extraction as described (35Sambrook J.F. Maniatis T. Sambrook J.F. Molecular Cloning: A Laboratory Manual. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY1989: 431-432Google Scholar). The sequence of the oligonucleotide used in the priming assays was 5′-AGAGGGAGATTTAGATGAGATGATTGAGGATGGAGATGTTGATGGAGAGATGATGAATGATGAGATGAGGG-3′. Expression and Purification of gp32-A and gp32-B Mutant Proteins—The gp32-A-petIMPACT or gp32-B-petIMPACT plasmids were transformed into BL21(DE3) cells and grown (separately) in 20 ml of Luria broth (LB) overnight at 37 °C. The expression and purification of gp32-A and gp32-B were identical. The overnight cultures were diluted 100-fold into two 1-liter flasks of LB and grown at 37 °C to an A600 of 0.8. The cultures were then allowed to cool to 18 °C, and protein expression was induced with 0.2 mm of isopropyl 1-thio-β-d-galactopyranoside. After 16 h of shaking, cells were collected by centrifugation at 6000 × g and resuspended in 80 ml of chitin column binding buffer containing one protease inhibitor pellet (Roche Applied Science). Cells were lysed using sonication, and cell debris was pelleted at 25,000 × g. Cell-free extract was loaded onto a 5-ml chitin column and washed with 200 column volumes of chitin binding buffer. The chitin resin was then resuspended in binding buffer plus 75 mm β-mercaptoethanol and incubated for 48 h at 4 °C to facilitate intein-mediated cleavage. Following cleavage, gp32-A or gp32-B proteins were eluted, dialyzed into storage buffer, and analyzed for purity using SDS-PAGE. Protein concentrations were determined by measuring the absorbance at 280 nm using an extinction coefficient of 38690 m-1 cm-1 for both gp32-A and gp32-B. Standard Minicircle Replication Reactions and Measurement of Primer Utilization—Replication reactions were carried out in a standard replication buffer (25 mm Tris acetate (pH 7.8), 125 mm KOAc, and 10 mm Mg(OAc)2) at 37 °C. The standard conditions used to assemble and initiate the replication reaction consisted of the following: 100 nm minicircle substrate; 200 nm each of gp43, gp45 (as trimer), and gp44/62; 600 nm each of monomer gp41, gp61, and gp59; 0.5 μm of gp32; 100 μm each of CTP, GTP, and UTP; 2 mm ATP; 50 μm each of dATP, dGTP, dCTP, and dTTP, in a typical reaction volume of 12 μl. The reaction was allowed to proceed for 2.5 min followed by a 10-fold dilution into replication buffer containing gp43, gp44/62, gp45, gp61, gp59, ATP, CTP, UTP, and GTP at the standard concentrations. Wild-type or gp32 mutant was included in the dilution buffer at a concentration of 2 μm along with 25 μCi of [α-32P]dGTP or [α-32P]dCTP for visualization of leading or lagging strands, respectively. 20-μl aliquots were removed at the indicated time points and quenched with a half-volume of 0.5 m EDTA. The quenched reactions were analyzed with either DE81 filter binding assays or alkaline agarose gel electrophoresis (34Yang J. Trakselis M.A. Roccasecca R.M. Benkovic S.J. J. Biol. Chem. 2003; 278: 49828-49838Abstract Full Text Full Text PDF PubMed Scopus (28) Google Scholar). For the filter binding assay, an 8-μl sample of the quenched reaction aliquot was spotted onto DE81 filters and allowed to air dry for ∼5 min. The filters were then washed 10 times with 100 ml of 300 mm ammonium formate or until the wash had an insignificant level of radioactivity as determined by a hand-held Geiger counter. Following the ammonium formate washes, the filters were washed once with 100% EtOH and allowed to air-dry for 30 min. Once dried, the filters were placed in LSC vials with 5 ml of Ecoscint LSC mixture in each vial and counted with single channel liquid scintillation counting. For the analysis of replication products using alkaline-agarose gel electrophoresis, 10 μl of the quenched reaction aliquot was mixed with 10 μl of loading dye (50% glycerol and 0.1% Orange G) and loaded onto a 15 × 25-cm 1% agarose gel in 30 mm NaOH and 1 mm EDTA. The samples were run at 40 V for 24 h in 30 mm NaOH and 1 mm EDTA buffer. After running, the gels were removed from their trays and neutralized by soaking in 1× TBE buffer for 1 h at room temperature. The neutralized gels were then dried onto a sheet of DE81 paper using a stack of paper towels to facilitate gel drying. After 4 h of drying with the paper towels, the gel and filter sheet were vacuum-dried. The dried gel and DE81 filter paper were then exposed overnight to a PhosphorImager plate and analyzed using a Storm 800 PhosphorImager system (Amersham Biosciences). To measure primer utilization, the standard replication reaction was performed by replacing [α-32P]dCTP with [α-32P]CTP in the dilution buffer. The reactions were quenched with an equal volume of 0.5 m EDTA at 2, 4, and 8 min after dilution. Reaction products were then mixed with loading dye (formamide with 0.2% xylene cyanol FF and bromphenol blue) and run on a 20% urea-acrylamide gel at 25 mA for 3 h at room temperature. The gel was then removed from the glass plates, wrapped in plastic film, and directly exposed to a PhosphorImager plate overnight. The PhosphorImager plate was analyzed using the Storm PhosphorImager. The total number of primers synthesized was calculated by summing the signal from the free and utilized primers. The percent primer utilization was determined by dividing the signal from utilized primers by the total number of primers. Single-stranded DNA Priming Assays—Priming reactions were carried out in the standard replication buffer containing 0.4 μm gp41, 0.4 μm gp61, 100 nm gp59 (ssM13 reactions only), 2 μm gp32 (wild-type or mutant where indicated), 2 mm ATP, 100 μm each of CTP, GTP, and UTP, and either 10 nm M13 ssDNA or 3 μm ssDNA oligonucleotide (strands) in a typical reaction volume of 25 μl. Approximately 20 μCi of [α-32P]CTP per reaction was used for labeling the primer. For dilution experiments using the primase trap protein, the reactions were initiated using the same protein, nucleotide, and ssM13 concentrations as above except that wt-gp32 was used exclusively in the pre-dilution reaction. The reactions were allowed to proceed for 2.5 min before a 10-fold dilution into buffer containing only nucleotides, trap protein (3 μm), and either wt-gp32 or gp32-A. The reactions were carried out at 37 °C, and aliquots were withdrawn at the indicated times and quenched with an equal volume of 0.5 m EDTA, pH 8.0. Priming products were analyzed by 20% urea-acrylamide sequencing gel electrophoresis. Autoradiography was accomplished as described above. Leading and Lagging Strand Replication with Wild-type and Mutant gp32 Proteins—The replisome was assembled using a low concentration wild-type gp32 (0.5 μm), followed by 10-fold dilution into replication buffer containing 2 μm wild-type or mutant gp32. This allows for the assembly and initiation of leading and lagging strand synthesis under normal conditions, followed by a rapid exchange of ssDNA-bound protein with the excess of protein in the dilution buffer (23Kadyrov F.A. Drake J.W. J. Biol. Chem. 2001; 276: 29559-29566Abstract Full Text Full Text PDF PubMed Scopus (34) Google Scholar). The minicircle DNA substrate was used so that the leading and lagging strand could be independently monitored using alkaline agarose gel electrophoresis or DE81 filter binding assays (Fig. 1). We examined the leading and lagging strand replication products with gp32-A, gp32-B, wt-gp32, and no gp32 in the dilution buffer (Fig. 2). Omission of gp32 from the dilution buffer leads to the inhibition of both leading and lagging strand DNA synthesis. This is a consequence of the rapid production of uncoated ssDNA that chelates the distributive replisomal proteins (e.g. gp45, gp44/62) and has been previously observed by us and others (15Salinas F. Benkovic S.J. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 7196-7201Crossref PubMed Scopus (54) Google Scholar, 36Kadyrov F.A. Drake J.W. Nucleic Acids Res. 2002; 30: 4387-4397Crossref PubMed Scopus (18) Google Scholar). Inclusion of gp32-A in the dilution buffer results in helicase-dependent leading strand DNA synthesis that is very similar to the reaction containing wt-gp32 in the dilution buffer. However, when compared with the wt-gp32 reaction, there is a significant increase in the amount of helicase-independent strand displacement synthesis (indicated by the black arrows in Fig. 2). This is likely because of a reduction in the ability of gp59 to "lock" the polymerase in an inhibited state in the absence of a gp59-gp32 interaction (37Lefebvre S.D. Wong M.L. Morrical S.W. J. Biol. Chem. 1999; 274: 22830-22838Abstract Full Text Full Text PDF PubMed Scopus (38) Google Scholar). Lagging strand DNA synthesis in the presence of gp32-A is reduced compared with the amount of leading strand synthesis produced in the same reaction, although the fragments tend to be longer. This indicates that the leading and lagging strand polymerases have become uncoupled (28Yang J. Nelson S.W. Benkovic S.J. Mol. Cell. 2006; 21: 153-164Abstract Full Text Full Text PDF PubMed Scopus (50) Google Scholar). Leading and lagging strand replication in the presence of gp32-B protein is reduced in a manner very similar to the reaction performed with gp32 omission. Presumably, the loss of cooperativity in ssDNA binding reduces the overall affinity to a level where gp32-B does not bind to ssDNA. For this reason, we focused our efforts at understanding the effects of gp32-A on lagging strand synthesis. We next examined the lagging strand products of reactions carried out with wt-gp32 or gp32-A in the dilution buffer in more detail. To equalize the relative amounts of signal in the reactions, we increased the amount of α-32P by 5-fold in the gp32-A reaction (Fig. 3). This enables an accurate determination of both the average Okazaki fragment length and the size distribution. As shown, the average length of Okazaki fragments in the gp32-A reaction is 2.3 times longer than the wt-gp32 reaction, and the size distribution is much broader in the presence of gp32-A (0.1-8 kb) as compared with the wild-type enzyme (0.1-2 kb). The production of longer Okazaki fragments can be caused by a decrease in either the primer synthesis rate or in the efficiency of primer handoff, or both (28Yang J. Nelson S.W. Benkovic S.J. Mol. Cell. 2006; 21: 153-164Abstract Full Text Full Text PDF PubMed Scopus (50) Google Scholar). To determine what mechanism is responsible for the increase in Okazaki fragment length in the presence of gp32-A, we carried out full replisome replication assays in the presence of [α-32P]CTP to label RNA primers produced by the primase. The products of these reactions were analyzed using urea-PAGE and quantitated as shown in Fig. 4. This analysis reveals both a decrease in the total primers synthesized (Fig. 4A) and in the percentage of primers being used for Okazaki fragment initiation by gp32-A (Fig. 4B). The decrease in primer number is not the result of fewer replication forks or a decrease in the amount of lagging strand template produced because the assembly of the replisome is carried out under identical conditions for both gp32-A and wt-gp32 reactions, and leading strand DNA synthesis is not affected by the gp32-A mutant (Fig. 2). The decrease in total primer synthesis could be the result of a reduction in catalytic efficiency of the primase protein itself (presumably through the loss of interaction with gp32) or a reduction in the number of replisomes containing primase (i.e. a decrease in binding rate or increase in dissociation rate). To determine whether gp32 directly affects the catalytic activity of the primase protein, we examined the activity of the primase using an ssDNA oligonucleotide containing a single priming recognition site. Under the conditions of this assay, the formation of a helicase-primase-DNA complex is efficient, and thus the overall rate of primer synthesis depends on the activity of the primase protein itself (i.e. the kcat for the priming reaction). Additionally, the loading of primase and helicase onto the ssDNA oligonucleotide does not require gp59 and therefore does not require a specific gp32-gp59 interaction. Compared with a reaction performed without gp32, the rate of primer synthesis is unaffected by the presence of either wt-gp32 or gp32-A (Fig. 4C). This indicates that the decrease in the priming rate of the gp32-A-containing replisome is not because of a reduc
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