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The Application of a Minicircle Substrate in the Study of the Coordinated T4 DNA Replication

2003; Elsevier BV; Volume: 278; Issue: 50 Linguagem: Inglês

10.1074/jbc.m307406200

ISSN

1083-351X

Autores

Jingsong Yang, Michael A. Trakselis, Rosa Maria Roccasecca, Stephen J. Benkovic,

Tópico(s)

Bacteriophages and microbial interactions

Resumo

A reconstituted in vitro bacteriophage T4 DNA replication system was studied on a synthetic 70-mer minicircle substrate. This substrate was designed so that dGMP and dCMP were exclusively incorporated into the leading and the lagging strand, respectively. This design allows the simultaneous and independent measurement of the leading and lagging strand synthesis. In this paper, we report our results on the characterization of the 70-mer minicircle substrate. We show here that the minicircle substrate supports coordinated leading and lagging strand synthesis under the experimental conditions employed. The rate of the leading strand fork movement was at an average of ∼150 nucleotides/s. This rate decreased to less than 30 nucleotides/s when the helicase was omitted from the reaction. These results suggest that both the holoenzyme and the primosome can be simultaneously assembled onto the minicircle substrate. The lagging strand synthesized on this substrate is of an average of 1.5 kb, and the length of the Okazaki fragments increased with decreasing [rNTPs]. The proper response of the Okazaki fragment size toward the change of the priming signal further indicates a functional replisome assembled on the minicircle template. The effects of various protein components on the leading and lagging strand synthesis were also studied. The collective results indicate that coordinated strand synthesis only takes place within certain protein concentration ranges. The optimal protein levels of the proteins that constitute the T4 replisome generally bracket the concentrations of the same proteins in vivo. Omission of the primase has little effect on the rate of dNMP incorporation or the rate of the fork movement on the leading strand within the first 30 s of the reaction. This inhibition only becomes significant at later times of the reaction and may be associated with the accumulation of single-stranded DNA leading to the collapse of active replisomes. A reconstituted in vitro bacteriophage T4 DNA replication system was studied on a synthetic 70-mer minicircle substrate. This substrate was designed so that dGMP and dCMP were exclusively incorporated into the leading and the lagging strand, respectively. This design allows the simultaneous and independent measurement of the leading and lagging strand synthesis. In this paper, we report our results on the characterization of the 70-mer minicircle substrate. We show here that the minicircle substrate supports coordinated leading and lagging strand synthesis under the experimental conditions employed. The rate of the leading strand fork movement was at an average of ∼150 nucleotides/s. This rate decreased to less than 30 nucleotides/s when the helicase was omitted from the reaction. These results suggest that both the holoenzyme and the primosome can be simultaneously assembled onto the minicircle substrate. The lagging strand synthesized on this substrate is of an average of 1.5 kb, and the length of the Okazaki fragments increased with decreasing [rNTPs]. The proper response of the Okazaki fragment size toward the change of the priming signal further indicates a functional replisome assembled on the minicircle template. The effects of various protein components on the leading and lagging strand synthesis were also studied. The collective results indicate that coordinated strand synthesis only takes place within certain protein concentration ranges. The optimal protein levels of the proteins that constitute the T4 replisome generally bracket the concentrations of the same proteins in vivo. Omission of the primase has little effect on the rate of dNMP incorporation or the rate of the fork movement on the leading strand within the first 30 s of the reaction. This inhibition only becomes significant at later times of the reaction and may be associated with the accumulation of single-stranded DNA leading to the collapse of active replisomes. The bacteriophage T4 DNA replication system has served as a successful working model for eukaryotic systems. A total of eight T4 proteins are required to reconstitute an in vitro replication fork that carries out efficient and coordinated leading and lagging strand synthesis (1.Benkovic S.J. Valentine A.M. Salinas F. Annu. Rev. Biochem. 2001; 70: 181-208Crossref PubMed Scopus (282) Google Scholar, 2.Nossal N.G. FASEB J. 1992; 6: 871-878Crossref PubMed Scopus (86) Google Scholar, 3.Alberts B.M. Philos. Trans. R. Soc. Lond-Biol. Sci. 1987; 317: 395-420Crossref PubMed Scopus (72) Google Scholar, 4.Nossal N.G. Karam J.D. Bacteriophage T4. 2nd Ed. American Society for Microbiology Press, Washington, D. C.1994: 43-53Google Scholar). These eight proteins form the T4 DNA replisome that can be subdivided into one primosome complex and two holoenzyme complexes based on their different functionalities. These subassemblies are believed to be structurally and functionally integrated through protein-protein interactions within the replisome. Three protein components are involved in the assembly of the holoenzyme complex: gp43 1The abbreviations used are: gpgene productTRFIItailed replicative form IIntnucleotidessDNAsingle-stranded DNALSCliquid scintillation countingrNTPribonucleoside triphosphate. (the polymerase), gp45 (the clamp protein), and gp44/62 (the clamp loader protein complex). The gp43 polymerase catalyzes the addition of a nucleotide monophosphate onto the 3′ end of the nascent DNA chain and also contains an editing 3′–5′ exonuclease activity (5.Hershfield M.S. Nossal N.G. J. Biol. Chem. 1972; 247: 3393-3404Abstract Full Text PDF PubMed Google Scholar). In the presence of the clamp protein, gp43 switches from a distributive to a processive enzyme (6.Capson T.L. Peliska J.A. Kaboord B.F. Frey M.W. Lively C. Dahlberg M. Benkovic S.J. Biochemistry. 1992; 31: 10984-10994Crossref PubMed Scopus (228) Google Scholar, 7.Huang C.C. Hearst J.E. Alberts B.M. J. Biol. Chem. 1981; 256: 4087-4094Abstract Full Text PDF PubMed Google Scholar, 8.Roth A.C. Nossal N.G. Englund P.T. J. Biol. Chem. 1982; 257: 1267-1273Abstract Full Text PDF PubMed Google Scholar). gp45 is a trimeric protein that forms a sliding clamp circumscribing the primer/template junction and increases the binding between gp43 and the DNA template (7.Huang C.C. Hearst J.E. Alberts B.M. J. Biol. Chem. 1981; 256: 4087-4094Abstract Full Text PDF PubMed Google Scholar). gp45 itself is loaded onto DNA by gp44/62, a molecular motor protein that utilizes energy from ATP hydrolysis for clamp loading (9.Kaboord B.F. Benkovic S.J. Curr. Biol. 1995; 5: 149-157Abstract Full Text Full Text PDF PubMed Scopus (66) Google Scholar, 10.Kaboord B.F. Benkovic S.J. Biochemistry. 1996; 35: 1084-1092Crossref PubMed Scopus (49) Google Scholar, 11.Trakselis M.A. Berdis A.J. Benkovic S.J. J. Mol. Biol. 2003; 326: 435-451Crossref PubMed Scopus (38) Google Scholar). The detailed kinetics of the multistep clamp loading process have been carefully studied by a series of pre-steady-state kinetics and stop-flow fluorescence resonance energy transfer experiments (12.Alley S.C. Abel-Santos E. Benkovic S.J. Biochemistry. 2000; 39: 3076-3090Crossref PubMed Scopus (53) Google Scholar, 13.Sexton D.J. Kaboord B.F. Berdis A.J. Carver T.E. Benkovic S.J. Biochemistry. 1998; 37: 7749-7756Crossref PubMed Scopus (48) Google Scholar, 14.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). gene product tailed replicative form II nucleotide single-stranded DNA liquid scintillation counting ribonucleoside triphosphate. The primosome complex constitutes another important component of the T4 replisome consisting of the helicase (gp41), the primase (gp61), and the helicase accessory protein (gp59). gp41 helicase forms a ring-shaped hexameric structure in the presence of ATP/GTP (15.Dong F. Gogol E.P. von Hippel P.H. J. Biol. Chem. 1995; 270: 7462-7473Abstract Full Text Full Text PDF PubMed Scopus (149) Google Scholar). This gp41 hexamer is assembled on the lagging strand and unwinds the double-strand DNA in front of the moving fork in the 5′ to 3′ direction (16.Liu C.C. Alberts B.M. J. Biol. Chem. 1981; 256: 2813-2820Abstract Full Text PDF PubMed Google Scholar, 17.Cha T.A. Alberts B.M. J. Biol. Chem. 1989; 264: 12220-12225Abstract Full Text PDF PubMed Google Scholar, 18.Raney K.D. Carver T.E. Benkovic S.J. J. Biol. Chem. 1996; 271: 14074-14801Abstract Full Text Full Text PDF PubMed Scopus (56) Google Scholar). In the presence of gp32, gp59 facilitates the loading of the helicase through its interactions with both proteins (19.Morrical S.W. Hempstead K. Morrical M.D. J. Biol. Chem. 1994; 269: 33069-33081Abstract Full Text PDF PubMed Google Scholar, 20.Morrical 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, 21.Lefebvre 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, 22.Ishmael F.T. Alley S.C. Benkovic S.J. J. Biol. Chem. 2001; 276: 25236-25242Abstract Full Text Full Text PDF PubMed Scopus (49) Google Scholar). gp61 is required to synthesize pentaribonucleotide primers for Okazaki fragment synthesis on the lagging strand. The recognition sequences for the T4 primase are 5′-GTT and 5′-GCT (23.Cha T.A. Alberts B.M. J. Biol. Chem. 1986; 261: 7001-7010Abstract Full Text PDF PubMed Google Scholar), with the former being the recognition site in vivo (24.Hinton D.M. Nossal N.G. J. Biol. Chem. 1987; 262: 10873-10878Abstract Full Text PDF PubMed Google Scholar, 25.Hinton D.M. Nossal N.G. J. Biol. Chem. 1985; 260: 12858-12865Abstract Full Text PDF PubMed Google Scholar). The primase activity is modulated by the presence of other proteins. For example, gp61 activity is greatly stimulated by the gp41 helicase (24.Hinton D.M. Nossal N.G. J. Biol. Chem. 1987; 262: 10873-10878Abstract Full Text PDF PubMed Google Scholar) and is further enhanced by the presence of both gp59 and gp32 (26.Valentine A.M. Ishmael F.T. Shier V.K. Benkovic S.J. Biochemistry. 2001; 40: 15074-15085Crossref PubMed Scopus (47) Google Scholar). Recently, strong evidence for the formation of a hexameric ring structure of gp61 within the primosome has been provided through both kinetic and biophysical studies (26.Valentine A.M. Ishmael F.T. Shier V.K. Benkovic S.J. Biochemistry. 2001; 40: 15074-15085Crossref PubMed Scopus (47) Google Scholar). The single-stranded DNA-binding protein, gp32, is an important component of the T4 replisome. It exhibits strong cooperative single-stranded DNA binding ability and is presumably functioning in stabilizing the loop structure formed during replication (27.Alberts B.M. Sternglanz R. Nature. 1977; 269: 655-661Crossref PubMed Scopus (114) Google Scholar). gp32 also interacts with a number of other T4 replication proteins in the replisome including gp59 and gp61 (22.Ishmael F.T. Alley S.C. Benkovic S.J. J. Biol. Chem. 2001; 276: 25236-25242Abstract Full Text Full Text PDF PubMed Scopus (49) Google Scholar, 28.Formosa T. Burke R.L. Alberts B.M. Proc. Natl. Acad. Sci. U. S. A. 1983; 80: 2442-2446Crossref PubMed Scopus (100) Google Scholar, 29.Burke R.L. Alberts B M. Hosoda J. J. Biol. Chem. 1980; 255: 11484-11493Abstract Full Text PDF PubMed Google Scholar). Study of the C-terminal deletion mutant of gp32 suggested the role of gp32 in the coordination and stabilization of the T4 replisome (29.Burke R.L. Alberts B M. Hosoda J. J. Biol. Chem. 1980; 255: 11484-11493Abstract Full Text PDF PubMed Google Scholar). One proposed activity of gp32 during replication is to inhibit the random priming by the primase on the lagging strand so that the priming event is only possible when a primer is required for the synthesis of the Okazaki fragment (30.Cha T.A. Alberts B.M. Biochemistry. 1990; 29: 1791-1798Crossref PubMed Scopus (43) Google Scholar). In vivo DNA synthesis requires the coordinated action of all the functional units within the replisome. It was first suggested by Alberts and co-workers (31.Morris C.F. Sinha N.K. Alberts B.M. Proc. Natl. Acad. Sci. U. S. A. 1975; 72: 4800-4804Crossref PubMed Scopus (55) Google Scholar) that the two T4 holoenzyme complexes must be coupled during replication in order to explain how the tethered holoenzyme complexes carry out the synthesis of anti-parallel DNA double strands. Their trombone model was later adopted in the Escherichia coli (32.Wu C.A. Zechner E.L. Hughes Jr., A.J. Franden M.A. McHenry C.S. Marians K.J. J. Biol. Chem. 1992; 267: 4064-4073Abstract Full Text PDF PubMed Google Scholar) and T7 replication system (33.Debyser Z. Tabor S. Richardson C.C. Cell. 1994; 77: 157-166Abstract Full Text PDF PubMed Scopus (79) Google Scholar). Studies of the E. coli system have provided the most convincing evidence for the presence of asymmetric and dimeric holoenzyme complexes (34.Maki H. Maki S. Kornberg A. J. Biol. Chem. 1988; 263: 6570-6578Abstract Full Text PDF PubMed Google Scholar, 35.McHenry C.S. Biochim. Biophys. Acta. 1988; 951: 240-248Crossref PubMed Scopus (11) Google Scholar, 36.Yuzhakov A. Turner J. O'Donnell M. Cell. 1996; 86: 877-886Abstract Full Text Full Text PDF PubMed Scopus (159) Google Scholar). A τ subunit has been shown to interact with both polymerases within the E. coli replisome (37.Kim S. Dallmann H.G. McHenry C.S. Marians K.J. J. Biol. Chem. 1996; 271: 21406-21412Abstract Full Text Full Text PDF PubMed Scopus (94) Google Scholar). Although a direct physical link between the two polymerases has not been identified in the T7 system, dilution experiments did suggest that the lagging strand polymerase was highly processive and was recycled during repetitive cycles of the Okazaki fragment synthesis (38.Lee J. Chastain II, P.D. Griffith J.D. Richardson C.C. J. Mol. Biol. 2002; 316: 19-34Crossref PubMed Scopus (56) Google Scholar). Recent studies (39.Salinas F. Benkovic S.J. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 7196-7201Crossref PubMed Scopus (54) Google Scholar, 40.Ishmael F.T. Trakselis M.A. Benkovic S.J. J. Biol. Chem. 2003; 278: 3145-3152Abstract Full Text Full Text PDF PubMed Scopus (43) Google Scholar) likewise support a dimeric polymerase in the bacteriophage T4 system. Besides a link between the two polymerases, there must be a coupled movement between the holoenzyme complex and the primosome as well. An interaction between the C terminus of the gp4 helicase/primase and the polymerase was identified in the T7 system (41.Notarnicola S.M. Mulcahy H.L. Lee J. Richardson C.C. J. Biol. Chem. 1997; 272: 18425-18433Abstract Full Text Full Text PDF PubMed Scopus (80) Google Scholar). Cha and Alberts (17.Cha T.A. Alberts B.M. J. Biol. Chem. 1989; 264: 12220-12225Abstract Full Text PDF PubMed Google Scholar) demonstrated that the T4 holoenzyme could perform rapid and processive synthesis with only the helicase present. Dong et al. (42.Dong F. Weitzel S.E. von Hippel P.H. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 14456-14461Crossref PubMed Scopus (44) Google Scholar) further showed that in the presence of a macromolecular crowding agent, rapid and processive synthesis can be carried out with a T4 polymerase/helicase "two-protein" system. These results strongly suggested an interaction between the leading strand holoenzyme and components of the primosome. The T4 DNA replication system has been studied on a tailed replicative form II (TRFII) DNA template constructed on M13 ssDNA. Only recently was a minicircle substrate utilized for more quantitative analyses (39.Salinas F. Benkovic S.J. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 7196-7201Crossref PubMed Scopus (54) Google Scholar, 43.Lee J. Chastain II, P.D. Kusakabe T. Griffith J.D. Richardson C.C. Mol. Cell. 1998; 1: 1001-1010Abstract Full Text Full Text PDF PubMed Scopus (125) Google Scholar). Compared with the TRFII M13 template, the minicircle system offers a number of advantages. With the minicircle substrate, the manipulation of the DNA sequence becomes possible. The design of the 70-mer minicircle substrate in this study enables the strand-specific incorporation of dGMP and dCMP into the leading and lagging strands, respectively. Two priming sites ∼40 nucleotides apart on the lagging strand also are present in this substrate. Another advantage of using a small circular substrate is that the smaller size of the minicircle provides a higher fork number/nucleotides ratio. As a result, a high concentration of the replication forks can be achieved in the reaction mixture. This allows the replication reactions to be carried out with the fork concentrations that are near or surpass the protein concentrations. There are concerns, however, about using such a substrate. One of the potential problems with the 70-mer minicircle is that the small size of the substrate may impose steric constraints on the loading of all replisome components. In particular, the simultaneous loading of both the holoenzyme and the primosome may be hindered on a 70-mer DNA substrate (44.Kadyrov F.A. Drake J.W. Nucleic Acids Res. 2002; 30: 4387-4397Crossref PubMed Scopus (18) Google Scholar). Should this be the case, the coordination between leading and lagging strand synthesis would be disrupted. In this paper, we assess the feasibility of the minicircle substrate as a fork template for the study of the coordinated T4 DNA replication. We have monitored the replication fork movement under various experimental conditions. The rate of fork movement varied from the highest measured rate of 250 nt/s to an average rate around 150 nt/s. We have developed a dual-label method that enables us to quantify simultaneously the amount of leading and lagging strand synthesis in the same reaction mixture. This greatly reduces the experimental error associated with the study of coordinated synthesis in which leading and lagging strand synthesis is measured in separate reactions. Our results support the assembly of an intact and functional replisome on the 70-mer substrate that established coordinated strand synthesis and responded to the variation of individual replisome protein concentrations. The protein concentrations that support coordinated synthesis agree well with their in vivo concentrations. Furthermore, we studied the effect of blocking lagging strand synthesis on the leading strand. Preventing primer synthesis by omitting gp61 did not affect the rate of the leading strand synthesis at early reaction times, but the effect became significant at later reaction times. A possible role of the interaction between the accumulated ssDNA and T4 proteins such as gp32 is also discussed. In the accompanying paper (57.Trakselis 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), we examined the recruitment or recycling of various protein components at the replication fork by using both the minicircle and the TRFII M13 template. [α-32P]dGTP and [α-32P]dCTP were purchased from PerkinElmer Life Sciences. [8-3H]dGTP was purchased from ICN Pharmaceuticals, Inc. Unlabeled deoxynucleotides and ribonucleotides were purchased from Roche Applied Science. Ecoscint LSC mixture was purchased from National Diagnostics. A Beckman LS6800 liquid scintillation counter was used for all LSC counting. Rapid kinetics was performed on a KinTek Chemical Quench Flow RQF-3 instrument (KinTek Corp.). Bacteriophage T4 proteins, exonuclease-deficient gp43 (45.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 (113) Google Scholar), gp44/62 (46.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 (47.Nossal N.G. J. Biol. Chem. 1979; 254: 6026-6031Abstract Full Text PDF PubMed Google Scholar), gp32, 2S. Alley, unpublished protocol. gp41 (48.Ishmael F.T. Alley S.C. Benkovic S.J. J. Biol. Chem. 2002; 277: 20555-20562Abstract Full Text Full Text PDF PubMed Scopus (41) Google Scholar), gp61 (26.Valentine A.M. Ishmael F.T. Shier V.K. Benkovic S.J. Biochemistry. 2001; 40: 15074-15085Crossref PubMed Scopus (47) Google Scholar), and gp59 (22.Ishmael F.T. Alley S.C. Benkovic S.J. J. Biol. Chem. 2001; 276: 25236-25242Abstract Full Text Full Text PDF PubMed Scopus (49) Google Scholar), were purified as described previously. 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 Mg(OAc)2. The Minicircle Substrate—A linear 70-mer oligonucleotide (5′-CAC CAT AAC CTC CAC CCT CCC CAA TAT TCA CCA TCA ACC CTT CAC CTC ACT TCA CTC CAC TAT ACC ACT C-3′) was intramolecularly ligated under dilute conditions using a bridging 20-mer oligonucleotide (5′-GGT TAT GGT GGA GTG GTA TA-3′) in a manner described previously (39.Salinas F. Benkovic S.J. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 7196-7201Crossref PubMed Scopus (54) Google Scholar, 43.Lee J. Chastain II, P.D. Kusakabe T. Griffith J.D. Richardson C.C. Mol. Cell. 1998; 1: 1001-1010Abstract Full Text Full Text PDF PubMed Scopus (125) Google Scholar). The minicircle was then annealed with a partially complementary 109-mer strand (5′-AGA GGA TGA TAT GGA GGA GAG GTA TAG GAG AGA GAA GTA TGT GGA GGT TAT GGT GGA GTG GTA TAG TGG AGT GAA GTG AGG TGA AGG GTT GAT GGT GAA TAT TGG GGA G-3′) which provides the template strand for lagging strand synthesis (Fig. 1). The Standard Replication Conditions and the Filter Binding Assay— Replication reactions were carried out in a complex buffer containing 25 mm Tris acetate (pH 7.5), 125 mm KOAc, and 10 mm Mg(OAc)2. The standard replication conditions used in all minicircle reactions consisted of 100 nm minicircle substrate, 240 nm each of gp43, gp45 (as trimer), and gp44/62, 600 nm each of monomer gp41, gp61, and gp59, 4.5 μm of gp32, 100 μm each of CTP, GTP, and UTP, 2 mm ATP, 100 μm each of dATP, dGTP, dCTP, and dTTP, [8-3H]dGTP (6.9 Ci/mmol), and [α-32P]dCTP (3000 Ci/mmol), in a typical reaction volume of 100 μl. Unless otherwise noted, the DNA polymerase holoenzymes (gp43, gp45, and gp44/62) were first preincubated at 37 °C with the minicircle DNA template in the presence of 2 mm ATP for 30 s followed by the addition of the primosome (gp41, gp61, and gp59) and gp32 along with dNTPs, rNTPs, and 2 mm ATP. 10–20 μl aliquots were removed at various time points and quenched with an equal volume of 0.5 m EDTA. The quenched reaction aliquots were then spotted onto DE81 filter paper. All filter papers were allowed to air-dry and then washed in 300 mm ammonium formate buffer (pH 8.0) until no radioactivity was detected in the wash. The filter papers were washed twice with 95% ethanol and allowed to air-dry in a hood. The dried filter papers were placed in LSC vials with 5 ml of Ecoscint LSC mixture in each vial and counted with dual-channel liquid scintillation counting (channel settings are based on an arbitrary scale of 0–1000. Channel 1 is set for 0–400 and channel 2 for 400–1000). The specific activities of both tritium and 32P were calculated by direct counting of a known volume of the reaction mixture in LSC mixture. Control experiments showed that there was no quenching effect of 32P on the filter paper. However, there was a 70% quenching of tritium by the filter paper. This effect was corrected in the calculation of the tritium-specific activity. The spill-over of 32P radioactivity into channel 1 was determined to be 2.2% of the total 32P radioactivity. The amount of dNMP incorporation (cpm) in the leading and the lagging strand was calculated according to Equations 1 and 2,32P=100×B/97.8(Eq. 1) 3H=A-2.2×B/97.8(Eq. 2) where A and B are the counts in channel 1 and 2, respectively. The Rate of the Replication Fork Movement—Unless otherwise specified, replication reactions were carried out under the standard conditions with either [α-32P]dGTP (3000 Ci/mmol) or [α-32P]dCTP (3000 Ci/mmol). Aliquots of the reaction mixture were sampled at various time intervals and quenched with an equal volume of 0.5 m EDTA, pH 8.0. The DNA products were analyzed either through 0.8% alkaline-agarose gel electrophoresis (30 mm NaOH and 5 mm EDTA) or through denaturing 4% PAGE. At the end of the separation, the alkaline gels were neutralized with 1 liter of TBE buffer, dried onto Whatman DE81 filter paper at room temperature for 12 h, and then dried under vacuum at 55 °C for 1 h. Autoradiography was obtained using a Molecular Dynamics Storm 800 PhosphorImager system (Amersham Biosciences). The length of the DNA was determined using Quantity One Quantitation Software (Bio-Rad) and comparing it to 32P-labeled DNA markers. [rNTPs] Dilution Experiment—Standard DNA replication reactions were carried out at various rNTP concentrations (200, 50, and 12.5 μm). [α-32P]dCTP (3000 Ci/mmol) was included in the reactions for the detection of the lagging strand synthesis and was added 1 min after the initiation of the reaction. The reactions were allowed to proceed for another 3 min before being quenched in equal volume of 0.5 m EDTA, pH 8.0. Reaction products were analyzed by electrophoresis on a 0.8% alkaline-agarose gel, and the sizes of the Okazaki fragments were analyzed as described above. Pre-steady-state DNA Replication Reactions—Pre-steady-state reactions were performed using a KinTek rapid chemical quench-flow instrument. All concentrations are after initiating the reaction. The reaction buffer was as described above. One sample syringe contained 100 nm minicircle template, 4.5 μm gp32, 1 mm ATP, 100 μm dNTPs, [α-32P]dGTP (3000 Ci/mmol), with or without 600 nm gp61, and with or without 100 μm rNTPs. The other sample syringe contained 240 nm gp43, 240 nm gp45 (as trimer), 240 nm gp44/62, 600 nm gp41 (as monomer), and 600 nm gp59. All reaction mixtures were pre-equilibrated at 37 °C. The reactions were initiated by rapid mixing of the contents in two syringes. The replication reaction was allowed to proceed for various time lengths, after which it was stopped by rapid addition of quench solution (pH 8.0, 500 mm EDTA). Reaction aliquots were spotted onto DE81 filter paper for determining the amount of incorporated radioactivity or analyzed by electrophoresis for assessing the rate of the fork movement as described above. The Rate of the Fork Movement—One of the concerns with using a 70-mer minicircle (Fig. 1) substrate is whether it has sufficient space for the proper assembly of the replisome (44.Kadyrov F.A. Drake J.W. Nucleic Acids Res. 2002; 30: 4387-4397Crossref PubMed Scopus (18) Google Scholar). The rate of the leading strand fork movement is a useful parameter for assessing the replisome assembly. Based on earlier results about the structure and stoichiometry of the T4 replisome, we set the following standard reaction condition to study replication on the 70-mer minicircle substrate: 100 nm minicircle substrate, 240 nm each of the holoenzyme components (gp43, gp45 trimer, and gp44/62), 600 nm each of the primosome components (gp41, gp61, and gp59, monomer concentration), and 4.5 μm gp32 with the stoichiometry of DNA: gp43:gp45:gp44/62:gp41:gp61:gp59:gp32 of 1:2.4:2.4:2.4:6:6:6:45. The rate of the leading strand fork movement was measured under these conditions. Fig. 2A shows a gel of the leading strand fork movement (lanes 1–3). The highest fork rate measured was around 250 nt/s with a KinTek rapid quench instrument (data not shown). The average rate by manual mixing was 150 nt/s (Fig. 2A). The higher rate probably reflects the capture of earlier events by the rapid quench method. The average rate is similar to the fork rate observed on a TRFII M13 template synthesized in our laboratory (57.Trakselis 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). Taken together, it seems that the fork rate on a minicircle substrate approaches that observed on a larger circular substrate. Other published reports describing T4 DNA replication on a 70-mer minicircle template showed that the fork extended only at a rate of 50 nt/s. Moreover, this rate was not affected by the presence or absence of the primosome, indicating that the loading of the primosome onto the 70-mer minicircle was hampered (44.Kadyrov F.A. Drake J.W. Nucleic Acids Res. 2002; 30: 4387-4397Crossref PubMed Scopus (18) Google Scholar). We note that these reactions were carried out at lower concentrations of both the minicircle substrate and the T4 replication proteins. It therefore appears that for the 70-mer minicircle substrate, higher concentrations of both the minicircle and the replication proteins are needed to assemble the functional replisome. The need for higher protein component concentrations for the minicircle substrate suggests decreased protein/protein and protein/DNA affinities within the assembled replisome. In particular, the assembly of the primosome and its contacts with the holoenzymes might be altered. We next determined the effect of the primosome components on the rate of the fork movement to test for the capability of the minicircle substrate to accommodate both the holoenzyme and the primosome complexes. The rate of the fork movement was measured for the omission of either gp59 alone or both gp41 and gp59. The order of addition of proteins features the assembly of the holoenzyme prior to that of the primosome. If the size of the minicircle substrate is not big enough for loading both the holoenzyme and the primosome, one would expect that the latter would not assemble. This was clearly not the case under our experimental conditions. Omission of either gp59 or both gp41/gp59 markedly decreased the rate of the fork movement, as compared with the

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