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Role of Protein-Protein Interactions during Herpes Simplex Virus Type 1 Recombination-dependent Replication

2004; Elsevier BV; Volume: 279; Issue: 21 Linguagem: Inglês

10.1074/jbc.m400832200

1083-351X

Amitabh V. Nimonkar, Paul E. Boehmer,

DNA Repair Mechanisms

Recombination-dependent replication is an integral part of the process by which double-strand DNA breaks are repaired to maintain genome integrity. It also serves as a means to replicate genomic termini. We reported previously on the reconstitution of a recombination-dependent replication system using purified herpes simplex virus type 1 proteins (Nimonkar A. V., and Boehmer, P. E. (2003) Proc. Natl. Acad. Sci. U. S. A. 100, 10201–10206). In this system, homologous pairing by the viral single-strand DNA-binding protein (ICP8) is coupled to DNA synthesis by the viral DNA polymerase and helicase-primase in the presence of a DNA-relaxing enzyme. Here we show that DNA synthesis in this system is dependent on the viral polymerase processivity factor (UL42). Moreover, although DNA synthesis is strictly dependent on topoisomerase I, it is only stimulated by the viral helicase in a manner that requires the helicase-loading protein (UL8). Furthermore, we have examined the dependence of DNA synthesis in the viral system on species-specific protein-protein interactions. Optimal DNA synthesis was observed with the herpes simplex virus type 1 replication proteins, ICP8, DNA polymerase (UL30/UL42), and helicase-primase (UL5/UL52/UL8). Interestingly, substitution of each component with functional homologues from other systems for the most part did not drastically impede DNA synthesis. In contrast, recombination-dependent replication promoted by the bacteriophage T7 replisome was disrupted by substitution with the replication proteins from herpes simplex virus type 1. These results show that although DNA synthesis performed by the T7 replisome is dependent on cognate protein-protein interactions, such interactions are less important in the herpes simplex virus replisome. Recombination-dependent replication is an integral part of the process by which double-strand DNA breaks are repaired to maintain genome integrity. It also serves as a means to replicate genomic termini. We reported previously on the reconstitution of a recombination-dependent replication system using purified herpes simplex virus type 1 proteins (Nimonkar A. V., and Boehmer, P. E. (2003) Proc. Natl. Acad. Sci. U. S. A. 100, 10201–10206). In this system, homologous pairing by the viral single-strand DNA-binding protein (ICP8) is coupled to DNA synthesis by the viral DNA polymerase and helicase-primase in the presence of a DNA-relaxing enzyme. Here we show that DNA synthesis in this system is dependent on the viral polymerase processivity factor (UL42). Moreover, although DNA synthesis is strictly dependent on topoisomerase I, it is only stimulated by the viral helicase in a manner that requires the helicase-loading protein (UL8). Furthermore, we have examined the dependence of DNA synthesis in the viral system on species-specific protein-protein interactions. Optimal DNA synthesis was observed with the herpes simplex virus type 1 replication proteins, ICP8, DNA polymerase (UL30/UL42), and helicase-primase (UL5/UL52/UL8). Interestingly, substitution of each component with functional homologues from other systems for the most part did not drastically impede DNA synthesis. In contrast, recombination-dependent replication promoted by the bacteriophage T7 replisome was disrupted by substitution with the replication proteins from herpes simplex virus type 1. These results show that although DNA synthesis performed by the T7 replisome is dependent on cognate protein-protein interactions, such interactions are less important in the herpes simplex virus replisome. Herpes simplex virus type 1 (HSV-1) 1The abbreviations used are: HSV-1, herpes simplex virus, type 1; D-loop, displacement loop; E-SSB, E. coli single-strand DNA-binding protein; pol, DNA polymerase; RDR, recombination-dependent replication; SDS, strand displacement synthesis; SSB, single-strand DNA-binding protein; ss, single-stranded; SV40, simian virus 40; TAg, large T antigen; Topo, topoisomerase; BSA, bovine serum albumin; nt, nucleotides; DTT, dithiothreitol; Dda, DNA-dependent ATPase; DSB, double-strand DNA breaks. 1The abbreviations used are: HSV-1, herpes simplex virus, type 1; D-loop, displacement loop; E-SSB, E. coli single-strand DNA-binding protein; pol, DNA polymerase; RDR, recombination-dependent replication; SDS, strand displacement synthesis; SSB, single-strand DNA-binding protein; ss, single-stranded; SV40, simian virus 40; TAg, large T antigen; Topo, topoisomerase; BSA, bovine serum albumin; nt, nucleotides; DTT, dithiothreitol; Dda, DNA-dependent ATPase; DSB, double-strand DNA breaks. is an ∼152-kbp double-stranded DNA virus (1Boehmer P.E. Villani G. Prog. Nucleic Acids Res. Mol. Biol. 2003; 75: 139-171Crossref PubMed Scopus (11) Google Scholar). Replication of the viral genome occurs in the nuclei of infected cells and requires at least seven virus-encoded proteins as well as several cellular factors (reviewed in Refs. 1Boehmer P.E. Villani G. Prog. Nucleic Acids Res. Mol. Biol. 2003; 75: 139-171Crossref PubMed Scopus (11) Google Scholar and 2Boehmer P.E. Nimonkar A.V. IUBMB Life. 2003; 55: 13-22Crossref PubMed Scopus (73) Google Scholar). The viral genome possesses distinct origins of replication and encodes an origin binding protein (UL9), which is highly suggestive of an initial θ-mode of replication (1Boehmer P.E. Villani G. Prog. Nucleic Acids Res. Mol. Biol. 2003; 75: 139-171Crossref PubMed Scopus (11) Google Scholar). Furthermore, the failure to detect genomic DNA ends shortly after infection hints at the possibility that the viral genome circularizes immediately upon infection and replicates via a θ-mode to generate circular intermediates (1Boehmer P.E. Villani G. Prog. Nucleic Acids Res. Mol. Biol. 2003; 75: 139-171Crossref PubMed Scopus (11) Google Scholar). The observation of high molecular weight viral DNA later during the life cycle (3Severini A. Scraba D.G. Tyrrell D.L. J. Virol. 1996; 70: 3169-3175Crossref PubMed Google Scholar) prompted the suggestion that replication switches to a rolling circle or σ-mode to produce head-to-tail concatamers that are subsequently cleaved into unit length genomes and packaged (2Boehmer P.E. Nimonkar A.V. IUBMB Life. 2003; 55: 13-22Crossref PubMed Scopus (73) Google Scholar). This strategy for replication, resembling that adopted by bacteriophage λ (4Enquist L.W. Skalka A. J. Mol. Biol. 1973; 75: 185-212Crossref PubMed Scopus (163) Google Scholar), has been considered the “dogma” for HSV-1 replication for more than 2 decades. However, recent evidence indicates that circularization of the genome is not a requisite for lytic viral replication and that the template for replication is in fact a linear genome (5Jackson S.A. DeLuca N.A. Proc. Natl. Acad. Sci. U. S. A. 2003; 100: 7871-7876Crossref PubMed Scopus (108) Google Scholar). Thus, it seems likely that the strategy of HSV-1 replication resembles more that of bacteriophages T4 and T7 (6Luder A. Mosig G. Proc. Natl. Acad. Sci. U. S. A. 1982; 79: 1101-1105Crossref PubMed Scopus (104) Google Scholar, 7Richardson C.C. Cell. 1983; 33: 315-317Abstract Full Text PDF PubMed Scopus (98) Google Scholar). Replication of a linear genome possesses an intrinsic problem, replication of the genomic termini. In T4, this is overcome by using recombination-dependent replication (RDR) in which the end of one genome invades into a homologous region of another and utilizes it as a template to complete lagging strand synthesis (6Luder A. Mosig G. Proc. Natl. Acad. Sci. U. S. A. 1982; 79: 1101-1105Crossref PubMed Scopus (104) Google Scholar). Such a mode of replication would generate highly branched replication intermediates that are commonly seen in HSV-1-infected cells (3Severini A. Scraba D.G. Tyrrell D.L. J. Virol. 1996; 70: 3169-3175Crossref PubMed Google Scholar).During the later stages of HSV-1 replication, the genome recombines at a high frequency, with a rate estimated to be 0.6%/kb of genome (8Smiley J.R. Wagner M.J. Summers W.P. Summers W.C. Virology. 1980; 102: 83-93Crossref PubMed Scopus (24) Google Scholar). In addition to recombination acting to replicate the ends of the linear genome, it may also act to repair double-strand DNA breaks (DSB). The HSV-1 genome possesses sites called a sequences that are recombination hotspots and are cleaved by endonuclease G to introduce DSB (9Wohlrab F. Chatterjee S. Wells R.D. Proc. Natl. Acad. Sci. U. S. A. 1991; 88: 6432-6436Crossref PubMed Scopus (22) Google Scholar, 10Huang K.J. Zemelman B.V. Lehman I.R. J. Biol. Chem. 2002; 277: 21071-21079Abstract Full Text Full Text PDF PubMed Scopus (30) Google Scholar). The a sequence-mediated cleavage of the genome is thought to be involved in genome isomerization (10Huang K.J. Zemelman B.V. Lehman I.R. J. Biol. Chem. 2002; 277: 21071-21079Abstract Full Text Full Text PDF PubMed Scopus (30) Google Scholar). DSB may also be generated as a consequence of oxidative damage induced upon infection (11Valyi-Nagy T. Olson S.J. Valyi-Nagy K. Montine T.J. Dermody T.S. Virology. 2000; 278: 309-321Crossref PubMed Scopus (69) Google Scholar, 12Milatovic D. Zhang Y. Olson S.J. Montine K.S. Roberts II, L.J. Morrow J.D. Montine T.J. Dermody T.S. Valyi-Nagy T. J. Neurovirol. 2002; 8: 295-305Crossref PubMed Scopus (45) Google Scholar).HSV-1 encodes its own replication machinery that consists of a single-strand DNA-binding protein (SSB) (ICP8), DNA helicase-primase (UL5/UL52 core enzyme and UL8 loading protein), and DNA polymerase (pol) (UL30 catalytic subunit and UL42 processivity factor) (2Boehmer P.E. Nimonkar A.V. IUBMB Life. 2003; 55: 13-22Crossref PubMed Scopus (73) Google Scholar). These factors have been shown to associate into a replisome that is capable of long chain leading and lagging strand synthesis (13Skaliter R. Lehman I.R. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 10665-10669Crossref PubMed Scopus (57) Google Scholar, 14Rabkin S.D. Hanlon B. J. Virol. 1990; 64: 4957-4967Crossref PubMed Google Scholar, 15Falkenberg M. Lehman I.R. Elias P. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 3896-3900Crossref PubMed Scopus (51) Google Scholar). We recently proposed a model for RDR in HSV-1 based on the ability of HSV-1-encoded factors to catalyze such reactions in vitro (16Nimonkar A.V. Boehmer P.E. Proc. Natl. Acad. Sci. U. S. A. 2003; 100: 10201-10206Crossref PubMed Scopus (41) Google Scholar). In our model, processing of DSB allows ICP8 to pair single-stranded (ss) donor DNA with complementary duplex DNA resulting in the formation of displacement loops (D-loops). These D-loops nucleate the assembly of the viral replisome that promotes long chain DNA synthesis in the presence of a DNA-relaxing enzyme (16Nimonkar A.V. Boehmer P.E. Proc. Natl. Acad. Sci. U. S. A. 2003; 100: 10201-10206Crossref PubMed Scopus (41) Google Scholar). A schematic representation of the reaction is shown in Fig. 1A.In this work, the ability of the HSV-1 replication proteins to promote DNA synthesis on D-loops (i.e. RDR) was examined in greater detail. Furthermore, we investigated the requirement for species-specific protein-protein interactions during HSV-1 RDR by replacing the HSV-1 replication proteins with their functional counterparts from other systems. Our results indicate that HSV-1 RDR exhibits a less stringent requirement for species-specific protein-protein interactions when compared with T7 RDR.EXPERIMENTAL PROCEDURESEnzymes and Reagents—Escherichia coli SSB (E-SSB), T4 gene 32 protein (gp32), and calf thymus DNA topoisomerase I (Topo I) were purchased from U. S. Biochemical Corp. One unit of Topo I is that amount of enzyme that relaxes 0.5 μg of pBR322 in 30 min at 37 °C. The specific activity of Topo I was 16,949 units/mg. T4 polynucleotide kinase and proteinase K were purchased from New England Biolabs and Roche Applied Science, respectively. ICP8 (17Boehmer P.E. Lehman I.R. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 8444-8448Crossref PubMed Scopus (90) Google Scholar), UL9 (18Sampson D.A. Arana M.E. Boehmer P.E. J. Biol. Chem. 2000; 275: 2931-2937Abstract Full Text Full Text PDF PubMed Scopus (7) Google Scholar), UL5/UL52 core enzyme, and UL8 (19Tanguy Le Gac N. Villani G. Hoffmann J.S. Boehmer P.E. J. Biol. Chem. 1996; 271: 21645-21651Abstract Full Text Full Text PDF PubMed Scopus (51) Google Scholar) were purified as described previously. Their concentrations, expressed in moles of monomeric protein, were determined using extinction coefficients of 82,720, 89,220, 171,380, and 130,390 m–1 cm–1 at 280 nm, respectively, calculated from their predicted amino acid sequences (20Gill S.C. von Hippel P.H. Anal. Biochem. 1989; 182: 319-326Crossref PubMed Scopus (5022) Google Scholar). UL30 and UL42 were purified as described, and their concentrations, in moles of monomeric protein, were determined by the method of Bradford using BSA as a standard (21Boehmer P.E. Methods Enzymol. 1996; 275: 16-35Crossref PubMed Scopus (10) Google Scholar). The T7 replication proteins, namely gene 2.5 protein (gp2.5), gene 5 protein/thioredoxin (gp5/Th), and gene 4 protein (gp4) were a kind gift from Dr. Charles C. Richardson (Harvard Medical School, Boston). Human RP-A (hRP-A), simian virus 40 (SV40), large T antigen (TAg), T4 DNA-dependent ATPase (Dda), and E. coli pol III holoenzyme (containing β subunit) were kind gifts from Drs. Patrick Sung (Yale University, New Haven, CT), James A. Borowiec (New York University School of Medicine, New York), Kevin Raney (University of Arkansas for Medical Sciences, Little Rock, AR), and Arthur Kornberg (Stanford University, Stanford, CA) respectively. ATP (disodium salt) and [γ-32P]ATP (4,500 Ci/mmol) were purchased from Sigma and MP Biomedicals, respectively. Deoxyribonucleoside triphosphates (disodium salts) were purchased from Amersham Biosciences.Nucleic Acids—Oligodeoxyribonucleotide PB11 (100-mer) (22Boehmer P.E. Dodson M.S. Lehman I.R. J. Biol. Chem. 1993; 268: 1220-1225Abstract Full Text PDF PubMed Google Scholar), complementary to residues 379–478 of the minus strand of pUC18, was synthesized and gel-purified by Sigma-Genosys. Its concentration was determined by using an extinction coefficient of 939,208.1 m–1 cm–1 at 260 nm. PB11 was 5′-32P-labeled with T4 polynucleotide kinase and purified using Sephadex G-25 (fine) quick spin columns (Roche Applied Science). pUC18 form I was isolated from Brij58-lysed cells followed by anion exchange (Q high (Bio-Rad)) as described previously (23Nimonkar A.V. Boehmer P.E. Nucleic Acids Res. 2003; 31: 5275-5281Crossref PubMed Scopus (14) Google Scholar). The DNA was further treated with 0.1 m NaOH, followed by neutralization and extraction with the Promega Wizard Plus DNA purification system. All DNA concentrations are expressed in moles of molecules.D-loop Formation—PB11 (10.5 nm) was preincubated with ICP8 or gp2.5 (250 nm) on ice for 8 min in a buffer containing 25 mm Tris acetate, pH 7.5, 10 mm magnesium acetate, 1 mm DTT, and 100 μg/ml BSA. The pairing reaction was initiated by adding pUC18 form I DNA (3.5 nm), and incubation was continued for 30 min at 30 °C. D-loops were purified by extraction with the Promega Wizard DNA clean-up system followed by removal of excess unannealed oligonucleotide by gel filtration through Chroma Spin + TE-1000 columns (BD Biosciences).DNA Synthesis Using Pre-formed D-loops—DNA synthesis reactions with purified D-loops were performed in a buffer containing 16.7 mm Tris acetate, pH 7.5, 6.7 mm magnesium acetate, 0.66 mm DTT, 2.5 mm ATP, 500 μm each of dATP, dCTP, dGTP, and TTP, and 66 μg/ml BSA. Unless otherwise stated, purified D-loops (0.6 nm) were supplemented with mixture A (400 nm ICP8, 5 nm UL30, 5 nm UL42, 10 nm UL5/UL52, 30 nm UL8, and 2.5 units of Topo I) or mixture B (200 nm gp2.5, 5 nm T7 gp5/Th, 10 nm T7 gp4, and 2.5 units of Topo I). Reactions were incubated for 60 min at 30 °C and quenched by the addition of termination buffer (final concentration, 50 mm EDTA and 3 μg/μl proteinase K) followed by further incubation for 20 min. Reaction products were resolved through 1% agarose containing 50 mm NaOH and 1 mm EDTA at ∼2.25 V/cm for 10 h. Following electrophoresis the gels were dried onto DE81 chromatography paper (Whatman), analyzed, and quantitated by storage phosphor analysis with a Molecular Dynamics Storm 820 PhosphorImager (Amersham Biosciences).DNA Synthesis Coupled to D-loop Formation—D-loops were formed using 250 nm ICP8 or gp2.5, 3.5 nm pUC18 form I DNA, and 10.5 nm PB11 in a buffer containing 16.7 mm Tris acetate, pH 7.5, 6.7 mm magnesium acetate, 0.66 mm DTT, 2.5 mm ATP, 500 μm each of dATP, dCTP, dGTP, and TTP, and 66 μg/ml BSA. Unless otherwise stated, D-loop reactions were supplemented with HSV-1 proteins (final concentration, 166 nm ICP8, 5 nm UL30/UL42, 10 nm UL5/UL52, 30 nm UL8) or T7 proteins (final concentration, 166 nm gp2.5, 5 nm gp5/Th, 10 nm gp4) and 2.5 units of Topo I, incubated for 15 min at 30 °C, and quenched by the addition of termination buffer (final concentration, 50 mm EDTA and 3 μg/μl proteinase K) followed by further incubation for 20 min. The final concentration of plasmid DNA in the reaction was 2.33 nm of which ∼15% were D-loops. The reaction products were resolved by two-dimensional native-denaturing agarose gel electrophoresis (16Nimonkar A.V. Boehmer P.E. Proc. Natl. Acad. Sci. U. S. A. 2003; 100: 10201-10206Crossref PubMed Scopus (41) Google Scholar). Each reaction was resolved in duplicate in the first dimension through 0.75% agarose-Tris acetate EDTA, pH 7.6, gels at 9 V/cm for 2 h. Under these conditions, free 100-mer migrates out of the gel. One of the lanes was used as a reference to visualize products resolved in the first dimension. The other lane was excised, soaked in buffer containing 50 mm NaOH and 1 mm EDTA, embedded in a second gel composed of 1% agarose in 50 mm NaOH and 1 mm EDTA, and electrophoresed at ∼2 V/cm for 7 h. Following electrophoresis the gels were dried onto DE81 chromatography paper (Whatman), analyzed, and quantitated by storage phosphor analysis with a Molecular Dynamics Storm 820 PhosphorImager (Amersham Biosciences).RESULTSLong Chain Synthesis during RDR Requires the UL42 Processivity Factor—We had demonstrated previously the requirement for the heterodimeric HSV-1 pol (UL30 and UL42) during RDR (16Nimonkar A.V. Boehmer P.E. Proc. Natl. Acad. Sci. U. S. A. 2003; 100: 10201-10206Crossref PubMed Scopus (41) Google Scholar). Here we examined the requirement for the viral processivity factor, UL42. Thus, purified D-loops were supplemented with increasing concentrations of UL30 or UL30/UL42, along with ICP8, helicase-primase, and Topo I. As shown in Fig. 1B, UL30 failed to generate full-length products (2686 nt) at concentrations ranging from 1 to 100 nm (lanes 1–6). Nevertheless, UL30 by itself could promote primer extension with increasing concentrations (Fig. 1B, lanes 3–6), leading to the formation of intermediates up to ∼1000 nt in length. On the other hand, the presence of equimolar UL42 enabled full-length synthesis (lanes 7–12), with complete products forming at UL30/UL42 concentrations as low as 5 nm (Fig. 1B, lane 9). Therefore, subsequent experiments were performed using UL30/UL42 at 5 nm.RDR Is Topo I-dependent and Stimulated by Helicase—We stated previously (16Nimonkar A.V. Boehmer P.E. Proc. Natl. Acad. Sci. U. S. A. 2003; 100: 10201-10206Crossref PubMed Scopus (41) Google Scholar) that efficient RDR is dependent on Topo I as well as helicase action. Here we examined the requirement for both proteins in more depth. Thus, purified D-loops were supplemented with increasing concentrations of helicase-primase in the absence and presence of Topo I, along with ICP8 and UL30/UL42. In the absence of Topo I, at helicase-primase concentrations ranging from 0 to 100 nm, only marginal DNA synthesis was observed (Fig. 2A, lanes 1–5). On the other hand, the presence of Topo I enabled full-length synthesis (Fig. 2A, lanes 6–10). Approximately 4% (∼33% of maximum) of the primer within the D-loop was extended to full length even in the absence of helicase-primase (Fig. 2A, lane 6). The presence of helicase-primase nevertheless stimulated the reaction ∼3-fold (Fig. 2B) and also increased the length of intermediates (Fig. 2A, compare lane 6 with lanes 7–10). Optimal RDR was observed with 2.5 units of Topo I (data not shown).Fig. 2RDR is Topo I-dependent and stimulated by helicase. Reactions were performed with purified D-loops and increasing UL5/UL52/UL8 concentrations, in the absence and presence of Topo I, as described under “Experimental Procedures,” followed by one-dimensional denaturing agarose gel electrophoresis. A, storage phosphorimage showing reaction products. Lanes 1–5, reactions in the absence of Topo I and 0, 12.5, 25, 50, and 100 nm helicase-primase, respectively; lanes 6–10, reactions in the presence of Topo I and 0, 12.5, 25, 50, and 100 nm helicase-primase, respectively. B, quantitation of full-length DNA synthesis as a function of UL5/UL52/UL8. Filled circles, reactions in the absence of Topo I; empty circles, reactions in the presence of Topo I. The positions of 100-mer, full-length (FL) extension products (2686 nt), intermediates, and markers (HindIII-digested λ DNA) are as indicated.View Large Image Figure ViewerDownload (PPT)Fig. 3 examines the role of the helicase-primase loading factor, UL8, during RDR. The data show that synthesis of longer intermediates (>500 nt) in the presence of helicase-primase was dependent on UL8 (Fig. 3, lane 2). In its absence, only shorter intermediates (<500 nt), as observed with Topo I alone, were synthesized (Fig. 3, compare lanes 1 and 3). Similar observations were made at a variety of UL5/UL52 concentrations (data not shown). Based on these data, subsequent experiments were performed using 2.5 units of Topo I, 10 nm UL5/UL52, and 30 nm UL8.Fig. 3UL8 increases the efficiency of primer utilization by helicase-primase during RDR. Reactions were performed with purified D-loops as described under “Experimental Procedures,” followed by one-dimensional denaturing agarose gel electrophoresis. Lane 1, reaction in the absence of UL5/UL52 and UL8; lane 2, reaction in the presence of UL5/UL52 (10 nm) and UL8 (30 nm); lane 3, reaction in the presence of UL5/UL52 (10 nm). The positions of 100-mer, full-length (FL) extension products (2686 nt), intermediates, and markers (HindIII-digested λ DNA) are as indicated. The three reactions were performed, electrophoresed simultaneously, and cropped from the same image.View Large Image Figure ViewerDownload (PPT)Importance of Species-specific Protein-Protein Interactions during RDR—The preceding experiments established the role of the various HSV-1 factors in our RDR system. To ascertain whether species-specific protein-protein interactions are important for efficient RDR, we performed a series of experiments wherein each of the HSV-1 replication components was replaced by its counterpart from a variety of systems. First, we substituted ICP8 with SSBs from other systems, namely hRP-A, E-SSB, T4 gp32, and T7 gp2.5. Fig. 4 depicts the results of this experiment. The concentrations of SSBs used in the experiment were twice that required to coat the D-loops, based on the site sizes of the SSBs on ssDNA (24Gourves A.S. Tanguy Le Gac N. Villani G. Boehmer P.E. Johnson N.P. J. Biol. Chem. 2000; 275: 10864-10869Abstract Full Text Full Text PDF PubMed Scopus (27) Google Scholar, 25Kim C. Paulus B.F. Wold M.S. Biochemistry. 1994; 33: 14197-14206Crossref PubMed Scopus (196) Google Scholar, 26Bujalowski W. Lohman T.M. Biochemistry. 1986; 25: 7799-7802Crossref PubMed Scopus (161) Google Scholar, 27Jensen D.E. Kelly R.C. von Hippel P.H. J. Biol. Chem. 1976; 251: 7215-7228Abstract Full Text PDF PubMed Google Scholar, 28Kim Y.T. Tabor S. Bortner C. Griffith J.D. Richardson C.C. J. Biol. Chem. 1992; 267: 15022-15031Abstract Full Text PDF PubMed Google Scholar). In the absence of SSB, most replication products accumulated as intermediates (<500 nt) and less than ∼1% of the primer reached full length (Fig. 4, lane 1). In the presence of ICP8, ∼15% of the primer was extended to full length (Fig. 4, lane 2). Substitution of ICP8 with hRP-A, E-SSB, and T4 gp32 generated full-length DNA molecules with little difference in efficiency (Fig. 4, lanes 3–5), except for gp32 that only promoted full-length synthesis to approximately half the level seen with ICP8 (Fig. 4, compare lanes 2 and 5). However, there was an abundance of small intermediates (100–500 nt) with the heterologous SSBs compared with ICP8 (Fig. 4, compare lane 2 with lanes 3–5). Most interesting, substitution of ICP8 with T7 gp2.5 completely abolished full-length synthesis and only permitted synthesis of short (<250 nt) intermediates (Fig. 4, lane 6). It should be noted that gp2.5 was able to stimulate the extension of singly primed M13 ssDNA by the T7 pol (data not shown), indicating that the protein was active. Moreover, results similar to those described were obtained for the various SSBs at a variety of concentrations (data not shown).Fig. 4Heterologous SSBs with the exception of T7 gp2.5 can support HSV-1 RDR. Reactions were performed with purified D-loops as described under “Experimental Procedures,” followed by one-dimensional denaturing agarose gel electrophoresis. Where indicated, ICP8 was replaced with the following SSBs: hRP-A, E-SSB, T4 gp32, and T7 gp2.5. The concentrations of the SSBs were 2-fold in excess to those required to coat the D-loops present in the reaction (0.6 nm). Lane 1, no SSB; lane 2, 400 nm ICP8; lane 3, 150 nm hRP-A; lane 4, 150 nm E-SSB; lane 5, 600 nm T4 gp32; lane 6, 600 nm T7 gp2.5. The positions of 100-mer, full-length (FL) extension products (2686 nt), intermediates, and markers (HindIII-digested λ DNA) are as indicated. The % full-length extension products are shown below the lane numbers.View Large Image Figure ViewerDownload (PPT)We then tested the effect of substituting the 5′–3′ unwinding activity of the HSV-1 helicase-primase with other helicases of like and opposing polarities. The helicases used in this experiment were the 5′–3′ helicases T4 Dda and T7 gp4 and the 3′–5′ helicases SV40 TAg and HSV-1 UL9, all of which have known functions in replication (29Jongeneel C.V. Bedinger P. Alberts B.M. J. Biol. Chem. 1984; 259: 12933-12938Abstract Full Text PDF PubMed Google Scholar, 30Park K. Debyser Z. Tabor S. Richardson C.C. Griffith J.D. J. Biol. Chem. 1998; 273: 5260-5270Abstract Full Text Full Text PDF PubMed Scopus (51) Google Scholar, 31Fairman M. Prelich G. Tsurimoto T. Stillman B. Biochim. Biophys. Acta. 1988; 951: 382-387Crossref PubMed Scopus (23) Google Scholar, 32Makhov A.M. Lee S.S. Lehman I.R. Griffith J.D. Proc. Natl. Acad. Sci. U. S. A. 2003; 100: 898-903Crossref PubMed Scopus (27) Google Scholar). The concentrations of all the helicases used in this experiment exhibited the same activity in unwinding a 100-mer annealed to M13 ssDNA (data not shown). Consistent with the observation made in Figs. 2 and 3, full-length products were generated in the absence of helicase (Fig. 5, lane 1), albeit less efficiently. The most striking effect was obtained upon replacing helicase-primase with Dda, where substitution prevented full-length synthesis, i.e. inhibited the helicase-independent extension reaction, and only led to limited primer extension (Fig. 5, compare lanes 1 and 2). Control reactions showed that this concentration of Dda efficiently unwound a 100-mer annealed to M13 ssDNA (data not shown). Fig. 5 also shows that substitution of helicase-primase (lane 4) with the other helicases, i.e. T7 gp4 (lane 3), TAg (lane 5), and UL9 (lane 6) decreased the efficiency of full-length DNA synthesis and led to the accumulation of short intermediates (<500 nt). Similar effects were observed at various concentrations of each helicase (data not shown).Fig. 5Heterologous helicases with the exception of T4 Dda can support HSV-1 RDR. Reactions were performed with purified D-loops as described under “Experimental Procedures,” followed by one-dimensional denaturing agarose gel electrophoresis. Where indicated, UL5/UL52/UL8 was replaced with the following helicases: Dda, T7 gp4, TAg, and UL9. Lane 1, no helicase; lane 2, Dda; lane 3, T7 gp4; lane 4, UL5/UL52/UL8; lane 5, TAg; lane 6, UL9. The concentration of each helicase was 12.5 nm. The positions of 100-mer, full-length (FL) extension products (2686 nt), intermediates, and markers (HindIII-digested λ DNA) are as indicated. The % full-length extension products are shown below the lane numbers.View Large Image Figure ViewerDownload (PPT)Finally, we studied the effect of replacing the dimeric HSV-1 pol (UL30/UL42) with heterologous pols. The enzymes tested were the replicative pols from T7 (gp5/Th) and E. coli (pol III). The concentration of the pols used was standardized by determining the amounts required to completely extend a 100-mer annealed to M13 ssDNA (in the presence of coating concentrations of T4 gp32 as SSB) (data not shown). In the absence of a pol, no extension was observed, thereby demonstrating the necessity for this activity during RDR (Fig. 6, lane 1). UL30/UL42 (Fig. 6, lane 2) as well as T7 gp5/Th (lane 3) were both capable of supporting RDR with comparable efficiency. E. coli pol III on the other hand only supported limited primer extension, generating products up to ∼200 nt and failing to generate full-length products (Fig. 6, lane 4). Similar results were obtained with higher concentrations of the pols (data not shown).Fig. 6T7 pol but not E. coli pol III can support HSV-1 RDR. Reactions were performed with purified D-loops as