Dissociative Properties of the Proteins within the Bacteriophage T4 Replisome
2003; Elsevier BV; Volume: 278; Issue: 50 Linguagem: Inglês
10.1074/jbc.m307405200
ISSN1083-351X
AutoresMichael A. Trakselis, Rosa Maria Roccasecca, Jingsong Yang, Ann M. Valentine, Stephen J. Benkovic,
Tópico(s)Protein Structure and Dynamics
ResumoDNA replication is a highly processive and efficient process that involves the coordination of at least eight proteins to form the replisome in bacteriophage T4. Replication of DNA occurs in the 5′ to 3′ direction resulting in continuous replication on the leading strand and discontinuous replication on the lagging strand. A key question is how a continuous and discontinuous replication process is coordinated. One solution is to avoid having the completion of one Okazaki fragment to signal the start of the next but instead to have a key step such as priming proceed in parallel to lagging strand replication. Such a mechanism requires protein elements of the replisome to readily dissociate during the replication process. Protein trapping experiments were performed to test for dissociation of the clamp loader and primase from an active replisome in vitro whose template was both a small synthetic DNA minicircle and a larger DNA substrate. The primase, clamp, and clamp loader are found to dissociate from the replisome and are continuously recruited from solution. The effect of varying protein concentrations (dilution) on the size of Okazaki fragments supported the protein trapping results. These findings are in accord with previous results for the accessory proteins but, importantly now, identify the primase as dissociating from an active replisome. The recruitment of the primase from solution during DNA synthesis has also been found for Escherichia coli but not bacteriophage T7. The implications of these results for RNA priming and extension during the repetitive synthesis of Okazaki fragments are discussed. DNA replication is a highly processive and efficient process that involves the coordination of at least eight proteins to form the replisome in bacteriophage T4. Replication of DNA occurs in the 5′ to 3′ direction resulting in continuous replication on the leading strand and discontinuous replication on the lagging strand. A key question is how a continuous and discontinuous replication process is coordinated. One solution is to avoid having the completion of one Okazaki fragment to signal the start of the next but instead to have a key step such as priming proceed in parallel to lagging strand replication. Such a mechanism requires protein elements of the replisome to readily dissociate during the replication process. Protein trapping experiments were performed to test for dissociation of the clamp loader and primase from an active replisome in vitro whose template was both a small synthetic DNA minicircle and a larger DNA substrate. The primase, clamp, and clamp loader are found to dissociate from the replisome and are continuously recruited from solution. The effect of varying protein concentrations (dilution) on the size of Okazaki fragments supported the protein trapping results. These findings are in accord with previous results for the accessory proteins but, importantly now, identify the primase as dissociating from an active replisome. The recruitment of the primase from solution during DNA synthesis has also been found for Escherichia coli but not bacteriophage T7. The implications of these results for RNA priming and extension during the repetitive synthesis of Okazaki fragments are discussed. The replisome of bacteriophage T4 serves as a model system to examine the intricate mechanisms of DNA replication. The eight proteins that comprise the T4 replisome are all functionally conserved in higher organisms. The foundation of the replisome is the DNA polymerase holoenzyme that is assembled from the polymerase (gp43), 1The abbreviations used are: gpgene productTRFIItailed replicative form IICPM7-diethylamino-3-(4′maleimidylphenyl)-4-methylcoumarinITCisothermal calorimetry. the clamp (gp45), and the clamp loader (gp44/62) proteins and catalyzes the processive incorporation of nucleotides in the 5′ to 3′ direction. Working in conjunction with the holoenzyme is the primosome, which is formed from the primase (gp61), the helicase (gp41), the helicase accessory protein (gp59), and single-stranded binding protein (gp32). Hexameric gp41 unwinds the double-strand DNA in an ATP- or GTP-dependent manner (1.Dong F. Gogol E.P. von Hippel P.H. J. Biol. Chem. 1995; 270: 7462-7473Abstract Full Text Full Text PDF PubMed Scopus (148) Google Scholar). gp32 acts to coat and protect the single-strand DNA generated by unwinding of the template DNA from degradation and to prevent reannealing. The hexameric primase synthesizes pentaribonucleotide primers needed for the initiation of each Okazaki fragment (2.Valentine A.M. Ishmael F.T. Shier V.K. Benkovic S.J. Biochemistry. 2001; 40: 15074-15085Crossref PubMed Scopus (46) Google Scholar). gp59, apparently acting as a hexamer, facilitates the assembly of the primosome complex (3.Ishmael F.T. Alley S.C. Benkovic S.J. J. Biol. Chem. 2002; 277: 20555-20562Abstract Full Text Full Text PDF PubMed Scopus (40) Google Scholar). The in vitro formation of a functional replisome provides an efficient replication machine amenable to detailed biochemical analyses. gene product tailed replicative form II 7-diethylamino-3-(4′maleimidylphenyl)-4-methylcoumarin isothermal calorimetry. Kinetic and biochemical analyses of individual proteins and protein subassemblies within this system have elucidated the structural and mechanistic details of parts of the replisome. The kinetic mechanism and the conformational changes that occur during the assembly of the DNA polymerase holoenzyme have been well characterized (4.Alley S.C. Abel-Santos E. Benkovic S.J. Biochemistry. 2000; 39: 3076-3090Crossref PubMed Scopus (53) Google Scholar, 5.Alley S.C. Trakselis M.A. Mayer M.U. Ishmael F.T. Jones A.D. Benkovic S.J. J. Biol. Chem. 2001; 276: 39340-39349Abstract Full Text Full Text PDF PubMed Scopus (27) Google Scholar, 6.Trakselis M.A. Alley S.C. Abel-Santos E. Benkovic S.J. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 8368-8375Crossref PubMed Scopus (100) Google Scholar, 7.Trakselis M.A. Berdis A.J. Benkovic S.J. J. Mol. Biol. 2003; 326: 435-451Crossref PubMed Scopus (38) Google Scholar). This assembly mechanism proceeds through 10 steps in a pathway that commences with the clamp loader binding to the clamp and hydrolyzing ATP along with many subsequent conformational changes. The final solution structure model of the holoenzyme complex has been depicted with the C terminus of gp43 inserted into a subunit interface of gp45 augmented by major protein contacts between the two (5.Alley S.C. Trakselis M.A. Mayer M.U. Ishmael F.T. Jones A.D. Benkovic S.J. J. Biol. Chem. 2001; 276: 39340-39349Abstract Full Text Full Text PDF PubMed Scopus (27) Google Scholar). Assembly of the primosome also proceeds through a number of steps to form its final complex consisting of several, apparently stacked hexameric ring structures. 2M. A. Trakselis and F. T. Ishmael, unpublished observations. The binding of gp59 to gp32 coated DNA facilitates the loading of a hexameric helicase with the lagging DNA strand passing through the center of a complex of the helicase and gp59 (3.Ishmael F.T. Alley S.C. Benkovic S.J. J. Biol. Chem. 2002; 277: 20555-20562Abstract Full Text Full Text PDF PubMed Scopus (40) Google Scholar). Monomeric gp59, which becomes hexameric in the presence of gp32, is thought to displace or destabilize gp32 from the single-strand DNA exposing a binding site for the helicase (8.Ishmael F.T. Alley S.C. Benkovic S.J. J. Biol. Chem. 2001; 276: 25236-25242Abstract Full Text Full Text PDF PubMed Scopus (48) Google Scholar, 9.Jones C.E. Mueser T.C. Dudas K.C. Kreuzer K.N. Nossal N.G. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 8312-8318Crossref PubMed Scopus (35) Google Scholar). The gp59 hexamer in turn stimulates formation of a hexameric gp41, and the latter in turn ultimately leads to the assembly of a hexameric primase (2.Valentine A.M. Ishmael F.T. Shier V.K. Benkovic S.J. Biochemistry. 2001; 40: 15074-15085Crossref PubMed Scopus (46) Google Scholar). All three proteins, gp59, gp41, gp61, are present in the initial primosome complex. Primosomes from other organisms are also thought to form multiprotein complexes. In bacteriophage T7, the primase and helicase functions are contained on one polypeptide, gp4, which has two domains: an N-terminal primase domain and a C-terminal helicase domain (10.Kusakabe T. Baradaran K. Lee J. Richardson C.C. EMBO J. 1998; 17: 1542-1552Crossref PubMed Scopus (31) Google Scholar, 11.Guo S. Tabor S. Richardson C.C. J. Biol. Chem. 1999; 274: 30303-30309Abstract Full Text Full Text PDF PubMed Scopus (81) Google Scholar, 12.Frick D.N. Baradaran K. Richardson C.C. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 7957-7962Crossref PubMed Scopus (66) Google Scholar). EM structures of gp4 show a hexameric protein complex that wraps around single-strand DNA forming a ring similar to what is seen with bacteriophage T4 3M. Norcum, unpublished observations. (13.Egelman H.H. Yu X. Wild R. Hingorani M.M. Patel S.S. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 3869-3873Crossref PubMed Scopus (252) Google Scholar). Only three additional proteins are required for coordinated leading and lagging strand synthesis in T7: the polymerase (gp5), single-stranded binding protein (gp2.5), and thioredoxin from the Escherichia coli host (14.Lee J. Chastain P.D. Kusakabe T. Griffith J.D. Richardson C.C. Mol. Cell. 1998; 1: 1001-1010Abstract Full Text Full Text PDF PubMed Scopus (124) Google Scholar). The change in the size of Okazaki fragments can be monitored to measure the effect of diluting certain proteins during active replication to determine their dissociative properties. Titrating any of the levels of protein components in T7 as well as varying the frequency of priming sites does not alter the Okazaki fragment size (14.Lee J. Chastain P.D. Kusakabe T. Griffith J.D. Richardson C.C. Mol. Cell. 1998; 1: 1001-1010Abstract Full Text Full Text PDF PubMed Scopus (124) Google Scholar). The lagging strand polymerase was found to recycle after completion of an Okazaki fragment, and all of the replisome proteins remained bound to the replication fork during the course of replication. It was suggested that the size of the Okazaki fragment was determined by the length of a replication loop and that a critical mass of bound gp2.5 was necessary to cause the recycling of the lagging strand polymerase after priming by gp4 (15.Lee J. Chastain P.D. Griffith J.D. Richardson C.C. J. Mol. Biol. 2002; 316: 19-34Crossref PubMed Scopus (56) Google Scholar). In E. coli, the rate of DNA synthesis is about three times faster than that of phage T4 or T7, perhaps reflecting its much greater genomic size and the number of proteins responsible for an active replisome. In the E. coli replisome, there are both a leading and lagging strand DNA pol III holoenzyme consisting of at least 10 gene products. The β protein acts as the clamp to hold the polymerases onto DNA, whereas τ acts to directly link the leading and lagging strand holoenzymes (16.O'Donnell M. Kuriyan J. Kong X.P. Stukenberg P.T. Onrust R. Mol. Biol. Cell. 1992; 3: 953-957Crossref PubMed Scopus (34) Google Scholar, 17.Studwell-Vaughan P.S. O'Donnell M. J. Biol. Chem. 1991; 266: 19833-19841Abstract Full Text PDF PubMed Google Scholar). The primosome consists of a helicase (DnaG), a primase (DnaB), and five other accessory proteins along with the single-stranded binding protein, SSB. By using dilution experiments, it was found that some proteins such as DnaB and β act distributively, whereas others such as DnaC and pol III act processively. Variation in the amount of dNTPs and rNTPs also changes the size of the Okazaki fragments (18.Wu C.A. Zechner E.L. McHenry C.S. Franden M.A. McHenry C.S. Marians K.J. J. Biol. Chem. 1992; 267: 4064-4073Abstract Full Text PDF PubMed Google Scholar). The mechanisms acting to control the size of the Okazaki fragments are not fixed at the time of assembly but can be varied during the lifetime of the replisome. Collectively, the data provide evidence for a highly interactive coordinated system where the discontinuous nature of lagging strand synthesis is orchestrated primarily by the primase dissociating/reassociating (recruitment from solution) with the replisome. Bacteriophage T4 has a replication system that shares properties with higher organisms, but the fewer number of protein components allows for more intense biochemical characterization. Coordinated DNA replication was also characterized in vitro for T4 with identical rates of leading and lagging strand synthesis on a minicircle template (19.Salinas F. Benkovic S.J. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 7196-7201Crossref PubMed Scopus (54) Google Scholar, 43.Yang J. Trakselis M.A. Roccasecca R.M. Benkovic S.J. J. Biol. Chem. 2003; 278: 49828-49838Abstract Full Text Full Text PDF PubMed Scopus (27) Google Scholar). Inhibition of the lagging strand synthesis by incorporation of ddCTP also inhibited the synthesis of the leading strand signifying coordination between the holoenzymes. Initial evidence to support the recycling of the polymerases was provided by Alberts et al. (20.Alberts 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) in studies where the concentration of gp43 did not change the size of the Okazaki fragments. Further dilution experiments on preformed replication complexes by Kadyrov and Drake (21.Kadyrov F.A. Drake J.W. J. Biol. Chem. 2001; 276: 29559-29566Abstract Full Text Full Text PDF PubMed Scopus (33) Google Scholar) found that the Okazaki fragment length increased upon dilution of gp32, gp45, and gp44/62 on a M13mp2 tailed double-stranded DNA substrate. These results thus identified the single-strand binding protein, the clamp, and clamp loader as acting distributively (dissociating/reassociating during replisome replication). Recently, Kadyrov and Drake (22.Kadyrov F.A. Drake J.W. Nucleic Acids Res. 2002; 30: 4387-4397Crossref PubMed Scopus (17) Google Scholar) extended their studies to examine the effect of using small minicircle substrates to study DNA replication and suggested that a small DNA substrate was unable to support coordinated replication. In the accompanying paper (43.Yang J. Trakselis M.A. Roccasecca R.M. Benkovic S.J. J. Biol. Chem. 2003; 278: 49828-49838Abstract Full Text Full Text PDF PubMed Scopus (27) Google Scholar), we showed that under our conditions the smaller 70-bp minicircle acts as a template for coordinated DNA synthesis and exhibits rates approaching that of the larger tailed replicative form II (TRFII) substrate derived from M13mp18. Therefore, utilization of this small minicircle DNA substrate under the appropriate conditions for biochemical characterization successfully mimics larger replication forks found in vivo. A complementary method to the previously reported dilution protocol uses protein traps to probe the dissociative properties of proteins within the replisome. Protein traps were utilized to verify the results obtained from dilution experiments and to identify additional protein components that may dissociate during replication with either the minicircle system or the TRFII template. Catalytically inactive mutants or dominant negatives of gp61 and gp44 were created to determine their effect when added in excess to a functioning replisome. Inhibition of DNA replication on either the leading or lagging strands by a protein trap addition suggests that the protein of interest dissociates from the replisome and is recruited from solution at some point during replication. Inactive mutants of gp44 and gp61 were found to inhibit minicircle DNA synthesis on both the lagging strand and the leading strand suggesting that the clamp loader and the primase dissociate from the replisome. Moreover, the addition of gp44 or gp61 protein traps to the TRFII DNA template increased the size of Okazaki fragments consistent with their dissociation from the replisome. These results are similar to findings in E. coli where the primase dissociates but differs from those for T7 where gp4 containing both priming and helicase activity does not dissociate. This study thus verified the dissociation/reassociation of the clamp loader and clamp proteins from an active T4 replisome but importantly identified the primase as also being continuously recruited to the replisome during DNA replication. [α-32P]dGTP and [α-32P]dCTP were purchased from PerkinElmer Life Sciences. Unlabeled deoxynucleotides and ribonucleotides were purchased from Roche Applied Science. 7-Diethylamino-3-(4′maleimidylphenyl)-4-methylcoumarin (CPM) was obtained from Molecular Probes (Eugene, OR). Bacteriophage T4 proteins, exonuclease-deficient gp43 (23.Frey M.W. Nossal N.G. Capson T.L. Benkovic S.J. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 2579-2583Crossref PubMed Scopus (111) Google Scholar), gp44/62 (24.Rush J. Lin T.C. Quinones M. Spicer E.K. Douglas I. Williams K.R. Konigsberg W.H. J. Biol. Chem. 1989; 264: 10943-10953Abstract Full Text PDF PubMed Google Scholar), gp45 (25.Nossal N.G. J. Biol. Chem. 1979; 254: 6026-6031Abstract Full Text PDF PubMed Google Scholar), gp32, 4S. Alley, unpublished observations. gp41 (3.Ishmael F.T. Alley S.C. Benkovic S.J. J. Biol. Chem. 2002; 277: 20555-20562Abstract Full Text Full Text PDF PubMed Scopus (40) Google Scholar), gp61 (2.Valentine A.M. Ishmael F.T. Shier V.K. Benkovic S.J. Biochemistry. 2001; 40: 15074-15085Crossref PubMed Scopus (46) Google Scholar), and gp59 (8.Ishmael F.T. Alley S.C. Benkovic S.J. J. Biol. Chem. 2001; 276: 25236-25242Abstract Full Text Full Text PDF PubMed Scopus (48) Google Scholar), were purified as described previously. Oligonucleotide primers and substrates were prepared as described previously (26.Alley S.C. Shier V.K. Abel-Santos E. Sexton D.J. Soumillion P. Benkovic S.J. Biochemistry. 1999; 38: 7696-7709Crossref PubMed Scopus (67) Google Scholar). The forked DNA template (Bio62/34/36-mer) was prepared previously (27.Kaboord B.F. Benkovic S.J. Curr. Biol. 1995; 5: 149-157Abstract Full Text Full Text PDF PubMed Scopus (65) Google Scholar). Deep Vent polymerase and all restriction enzymes were from New England Biolabs. All other chemicals were of analytical grade or better. The complex buffer used in all experiments was 25 mm Tris-OAc (pH 7.5), 150 mm KOAc, and 10 mm MgOAc. DNA Substrates—The 70/109-bp minicircle substrate was prepared (Fig. 1A) as described in the accompanying paper (43.Yang J. Trakselis M.A. Roccasecca R.M. Benkovic S.J. J. Biol. Chem. 2003; 278: 49828-49838Abstract Full Text Full Text PDF PubMed Scopus (27) Google Scholar). The TRFII DNA substrate was prepared (Fig. 1A) as described previously with few modifications (21.Kadyrov F.A. Drake J.W. J. Biol. Chem. 2001; 276: 29559-29566Abstract Full Text Full Text PDF PubMed Scopus (33) Google Scholar, 28.Mok M. Marians K.J. J. Biol. Chem. 1987; 262: 2304-2309Abstract Full Text PDF PubMed Google Scholar). DNA annealing was performed in a 40-μl volume of a buffer containing 20 mm Tris acetate (pH 7.5), 10 mm MgOAc, 1 mm dithiothreitol, 250 nm M13mp18 (as circular chromosomes), and 375 nm of a 73-mer oligonucleotide (5′-(T)43GATGTCCGCAACATCAAACATGACCACTGC-3′) whose 30 3′ nucleotides are complementary to the viral DNA, which was heated for 5 min at 95 °C and cooled at room temperature for 2 h. The annealed DNA was immediately used in a DNA synthesis reaction in a total volume of 1 ml of a buffer containing 20 mm Tris acetate (pH 7.5), 10 mm MgOAc, 1 mm dithiothreitol, 65 mm KOAc, 50 mm annealed DNA, 1 mm ATP, 0.2 mm dATP, 0.2 mm dGTP, 0.2 mm dCTP, 0.2 mm dTTP, 0.1 mg/ml bovine serum albumin, 34 μm gp43, 450 μm gp45 (as monomer), 148 μm gp44/62 and incubated for 50 min at 37 °C. The mixture was purified once by phenol/chloroform extraction, precipitated with ethanol, resuspended in TE buffer, and passed through micro-spin columns (Bio-Rad). The concentration of the substrate was determined by absorption at 260 nm. Neutral agarose gel electrophoresis of 1 μg of substrate revealed that 90% of single-strand DNA was converted into a tailed double-stranded form. Creation of Dominant Negative Proteins—The primase trap was created by site-directed mutagenesis to change a conserved glutamic acid (Glu-234) thought to be important for catalysis as identified by sequence alignments with other primases (29.Strack B. Lessl M. Calendar R. Lanka E. J. Biol. Chem. 1992; 267: 13062-13072Abstract Full Text PDF PubMed Google Scholar, 30.Aravind L. Leipe D.D. Koonin E.V. Nucleic Acids Res. 1998; 26: 4205-4213Crossref PubMed Scopus (311) Google Scholar). An E234Q mutant of gp61 was created using the QuickChange protocol (Stratagene) from WT pUC18-gp61 (2.Valentine A.M. Ishmael F.T. Shier V.K. Benkovic S.J. Biochemistry. 2001; 40: 15074-15085Crossref PubMed Scopus (46) Google Scholar). The following PCR primers were used in the preparation of gp61-E234Q which disrupted a native XbaI site: A, 5′-CGA GTT AAA GAT GGT GAT GTA TAT GTT CTA CAA GGA CC-3′; B, 5′-GGT CCT TGT AGA ACA TAT ACA TCA CCA TCT TTA ACT CG. pUC18-gp61(E234Q) was digested with SapI and NdeI and ligated into a similarly digested pETIMPACT vector to yield pETIMPACT-gp61(E234Q). Mutations were confirmed by DNA sequencing (Pennsylvania State University Nucleic Acid Facility). Purification of gp61-E234Q was performed as described previously (2.Valentine A.M. Ishmael F.T. Shier V.K. Benkovic S.J. Biochemistry. 2001; 40: 15074-15085Crossref PubMed Scopus (46) Google Scholar). The clamp loader trap was also created by site-directed mutagenesis by changing the conserved lysine in the nucleotide binding domain or p-loop of the Walker A motif of gp44 to affect ATP turnover. Cloning of gp44(K56A)/62 was performed using the following PCR primers: A, 5′-AGG AAC AGG TGC CAC AAC TGT AGC; B, 5′-ATA TCC ATG GTT ATC ACT TCC ACT GCA TTT CGC; C, 5′-AGG AAC AGG TGC CAC AAC TGT AGC; and D, 5′-AGA TTA CAT ATG ATT ACT GTA AAT GAA AAT GAA AAA GAA C. Fragment AD was prepared by PCR using primers A and D with pET26b-gp44 as template. Fragment BC was created similarly using PCR. Overlap extension PCR using fragments AD and BC yielded the mutant gp44-K56A. The full-length fragment was digested with NdeI and NcoI and ligated into similarly digested pET26b to yield pET26b-gp44(K56A). Mutations were confirmed by DNA sequencing (Pennsylvania State University Nucleic Acid Facility). pET26b-gp44(K56A) was cotransformed with pET22b-gp62 into BL21DE3 cells. The protein was induced with 0.2 mm isopropyl-1-thio-β-d-galactopyranoside at 25 °C for 5 h. Purification of gp44(K56A)/gp62 was performed as described previously (24.Rush J. Lin T.C. Quinones M. Spicer E.K. Douglas I. Williams K.R. Konigsberg W.H. J. Biol. Chem. 1989; 264: 10943-10953Abstract Full Text PDF PubMed Google Scholar). Activity Assays for the Dominant Negative Protein Traps—An ATPase assay was performed with gp44(K56A)/62 as described previously (26.Alley S.C. Shier V.K. Abel-Santos E. Sexton D.J. Soumillion P. Benkovic S.J. Biochemistry. 1999; 38: 7696-7709Crossref PubMed Scopus (67) Google Scholar) to test for its anticipated reduced specific activity. The inability of gp61(E234Q) to make pentaribonucleotide primers was verified with a priming assay as described previously (2.Valentine A.M. Ishmael F.T. Shier V.K. Benkovic S.J. Biochemistry. 2001; 40: 15074-15085Crossref PubMed Scopus (46) Google Scholar). Minicircle assays for DNA replication were also used to test the activity of the protein traps, gp44(K56A)/62 and gp61(E234Q). Binding Assays for the Dominant Negative Protein Traps—Steady-state fluorescence experiments were performed essentially as described on an ISA (Edison, NJ) FluoroMax-2 spectrofluorimeter thermostated to 25 °C (31.Alley S.C. Jones A.D. Soumillion P. Benkovic S.J. J. Biol. Chem. 1999; 274: 24485-24489Abstract Full Text Full Text PDF PubMed Scopus (25) Google Scholar). Fluorescence titrations were carried out to determine the KD value for the interaction between gp44(K56A)/62 with gp45(S158C/W199F/W92F)-CPM (6.Trakselis M.A. Alley S.C. Abel-Santos E. Benkovic S.J. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 8368-8375Crossref PubMed Scopus (100) Google Scholar). Excitation of the tryptophan donors in gp44(K46A)/62 at 280 nm was used to detect energy transfer to a CPM acceptor attached to gp45(S158C/W199F/W92F) devoid of tryptophans. Tryptophan quenching served to determine the binding curve. Slit widths were 2 nm, and emission was monitored in 1-nm increments with 0.5-s integration time. Isothermal titration calorimetry (ITC) experiments were performed on a thermostated MicroCal VP-ITC system as described previously (2.Valentine A.M. Ishmael F.T. Shier V.K. Benkovic S.J. Biochemistry. 2001; 40: 15074-15085Crossref PubMed Scopus (46) Google Scholar). gp61 and gp61(E234Q) were dialyzed into complex buffer, and the 45-mer containing a 5′-GTT priming site (5′-G GGT GGG AGG GAG GTT TGC AAC TGA TCG ATG ATA GTA CGT CTG TG) was diluted directly into complex buffer. For all experiments, the 45-mer was titrated into the sample cell containing either gp61 or gp61(E234Q), and the binding sites (N), dissociation constant (KD), and thermodynamic properties (ΔH and ΔS) were analyzed using Origin 5.0 (OriginLab, Northampton, MA). Replication Assays—The standard reaction conditions used in all minicircle reactions were described in detail in the accompanying paper (43.Yang J. Trakselis M.A. Roccasecca R.M. Benkovic S.J. J. Biol. Chem. 2003; 278: 49828-49838Abstract Full Text Full Text PDF PubMed Scopus (27) Google Scholar) and consisted of 100 nm minicircle, 200 nm holoenzyme (gp43, gp45 trimers, and gp44/62), 600 nm primosome (gp41, gp61, and gp59 as monomers), and 5 μm gp32 in complex buffer consisting of 20 mm Tris (pH 7.5), 150 mm KOAc, and 10 mm MgOAc, 500 μm each of dATP, dGTP, dCTP, and dTTP, 4 mm ATP, and 500 μm each of CTP, GTP, and UTP in a 100-μl total volume (Fig. 1B). [α-32P]dGTP (3000 Ci/mmol) and [α-32P]dCTP (3000 Ci/mmol) were present at 100 μCi/ml to measure leading and lagging strand synthesis, respectively. Reaction conditions with the TFRII DNA template were as follows: 3 nm TRFII, 16.6 nm gp43, 104 nm gp45 trimers, 16.2 nm gp44/62, 20 nm gp41 (hexamers), 36 nm gp61 (hexamers), 57 nm gp59 (hexamers), 4 μm gp32, 2 mm ATP, 200 μm dNTPs, 400 μm rNTPs, and 250 μCi/ml [α-32P]dGTP in complex buffer. Different orders of addition of the components were examined to determine the best substrate sequence for the reaction. Unless otherwise noted, the DNA polymerase holoenzyme (gp43, gp45, and gp44/62) was first preincubated at 37 °C with the DNA template in the presence of half of the ATP concentration for 1 min followed by the addition of the primosome (gp41, gp61, and gp59) and gp32 along with dNTPs, rNTPs, and the other half of ATP concentration (either 2 mm for the minicircle or 1 mm for TFRII). 5–20-μl aliquots were removed at various time points and quenched with an equal volume of 0.5 mm EDTA. Addition of a 5-fold molar excess of protein trap or additional protein components occurred at 90 s into the reaction for minicircle substrates and 5 min for TRFII substrates unless otherwise noted (Fig. 1C). To determine the size of leading and lagging strand products, samples were electrophoresed on a 0.8% alkaline agarose gel (30 mm NaOH and5mm EDTA) for 36 h. After electrophoresis, the gels were dried onto DE81 paper and visualized using a Molecular Dynamics PhosphorImager (Amersham Biosciences). Analysis of the distribution of DNA products on the alkaline gel through visualization by the PhosphorImager was quantified over the length of the lane. Determination of the amount of leading and lagging strand synthesis in the minicircle system was done by running the samples on a second 20% denaturing PAGE gel followed by PhosphorImaging. For the TFRII substrate quantification of the amount of leading and lagging strand synthesis was measured by PhosphorImager screening of two rectangles in each lane, one encircling the area above the 7249 bases (the length of the substrate), corresponding to the leading strand products, and the second one below the same band encompassing the lagging strand products. The specific activity of the 32P in the reaction was determined by spotting various amounts of the diluted reaction mixture on DE81 filter papers, in order to generate a standard curve whose slope was the specific activity of the radiolabeled nucleotide (counts/pmol dNTPs). This value was used to calculate the pools of dNTPs at each time point in the reaction to obtain the rate of pools dNTPs/min incorporated into either the leading or lagging strands. An average of at least four areas was used to calculate and correct for the background value of the gel. Concentration Titration Experiments—Different starting concentrations of either gp44/62, gp45, or gp61 were used in the minicircle reaction to determine the effect of concentration on Okazaki fragment size. The replisome was preassembled as described with either 4- or 16-fold lower concentrations of either gp44/62 or gp45, and 3- or 9-fold lower concentrations of gp61. [α-32P]dCTP was added to the reaction mixture 60 s after the start time in order to detect only Okazaki fragments made from active replisomes, and the reaction was then quenched with EDTA after 3 min. The different reaction mixtures were then electrophoresed on an alkaline agarose gel, and the sizes of the Okazaki fragments were analyzed. Data Analysis—Percent active replication complexes can be estimated using two different methods. The first method calculates the number of complexes that are active throughout the reaction cycle compared with a theoretical value, and the second method calculates the number of complexes that have been active. The theoretical value is based on the rate of DNA synthesis in bp/s and the concentration of the replisome (equivalent to the concentration of the mi
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