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The Herpes Simplex Virus Type-1 Single-strand DNA-binding Protein, ICP8, Increases the Processivity of the UL9 Protein DNA Helicase

1998; Elsevier BV; Volume: 273; Issue: 5 Linguagem: Inglês

10.1074/jbc.273.5.2676

ISSN

1083-351X

Autores

Paul E. Boehmer,

Tópico(s)

Vector-Borne Animal Diseases

Resumo

Herpes simplex virus type-1 UL9 protein is a sequence-specific DNA-binding protein that recognizes elements in the viral origins of DNA replication and possesses DNA helicase activity. It forms an essential complex with its cognate single-strand DNA-binding protein, ICP8. The DNA helicase activity of the UL9 protein is greatly stimulated as a consequence of this interaction. A complex of these two proteins is thought to be responsible for unwinding the viral origins of DNA replication. The aim of this study was to identify the mechanism by which ICP8 stimulates the translocation of the UL9 protein along DNA. The data show that the association of the UL9 protein with DNA substrate is slow and that its dissociation from the DNA substrate is fast, suggesting that it is nonprocessive. ICP8 caused maximal stimulation of DNA unwinding activity at equimolar UL9 protein concentrations, indicating that the active species is a complex that contains UL9 protein and ICP8 in 1:1 ratio. ICP8 prevented dissociation of UL9 protein from the DNA substrate, suggesting that it increases its processivity. ICP8 specifically stimulated the DNA-dependent ATPase activity of the UL9 protein with DNA cofactors that allow translocation of UL9 protein and those with secondary structure. These data suggest that UL9 protein and ICP8 form a specific complex that translocates along DNA. Within this complex, ICP8 tethers the UL9 protein to the DNA substrate, thereby preventing its dissociation, and participates directly in the assimilation and stabilization of the unwound DNA strand, thus facilitating translocation of the complex through regions of duplex DNA. Herpes simplex virus type-1 UL9 protein is a sequence-specific DNA-binding protein that recognizes elements in the viral origins of DNA replication and possesses DNA helicase activity. It forms an essential complex with its cognate single-strand DNA-binding protein, ICP8. The DNA helicase activity of the UL9 protein is greatly stimulated as a consequence of this interaction. A complex of these two proteins is thought to be responsible for unwinding the viral origins of DNA replication. The aim of this study was to identify the mechanism by which ICP8 stimulates the translocation of the UL9 protein along DNA. The data show that the association of the UL9 protein with DNA substrate is slow and that its dissociation from the DNA substrate is fast, suggesting that it is nonprocessive. ICP8 caused maximal stimulation of DNA unwinding activity at equimolar UL9 protein concentrations, indicating that the active species is a complex that contains UL9 protein and ICP8 in 1:1 ratio. ICP8 prevented dissociation of UL9 protein from the DNA substrate, suggesting that it increases its processivity. ICP8 specifically stimulated the DNA-dependent ATPase activity of the UL9 protein with DNA cofactors that allow translocation of UL9 protein and those with secondary structure. These data suggest that UL9 protein and ICP8 form a specific complex that translocates along DNA. Within this complex, ICP8 tethers the UL9 protein to the DNA substrate, thereby preventing its dissociation, and participates directly in the assimilation and stabilization of the unwound DNA strand, thus facilitating translocation of the complex through regions of duplex DNA. Herpes simplex virus type-1 (HSV-1) 1The abbreviations used are: HSV-1, herpes simplex virus type-1; SSB, single-strand DNA-binding protein; ssDNA, single-stranded DNA; E-SSB, E. coli SSB; EPPS, N-(2-hydroxyethyl)piperazine-N′-(3-propanesulfonic acid). 1The abbreviations used are: HSV-1, herpes simplex virus type-1; SSB, single-strand DNA-binding protein; ssDNA, single-stranded DNA; E-SSB, E. coli SSB; EPPS, N-(2-hydroxyethyl)piperazine-N′-(3-propanesulfonic acid). is a double-stranded DNA virus with a genome of ∼152 kilobase pairs that contains three origins of DNA replication (1Boehmer P.E. Lehman I.R. Annu. Rev. Biochem. 1997; 66: 347-384Crossref PubMed Scopus (264) Google Scholar). Replication of origin-containing plasmids requires the action of seven viral gene products (2Wu C.A. Nelson N.J. McGeoch D.J. Challberg M.D. J. Virol. 1988; 62: 435-443Crossref PubMed Google Scholar, 3McGeoch D.J. Dalrymple M.A. Dolan A. McNab D. Perry L.J. Taylor P. Challberg M.D. J. Virol. 1988; 62: 444-453Crossref PubMed Google Scholar). These seven gene products comprise a highly processive heterodimeric DNA polymerase (UL30/UL42 genes), a heterotrimeric DNA helicase-primase (UL5/UL8/UL52genes), a single-strand DNA-binding protein (SSB) (UL29gene), and an origin-binding protein (UL9 gene) (reviewed in Ref. 1Boehmer P.E. Lehman I.R. Annu. Rev. Biochem. 1997; 66: 347-384Crossref PubMed Scopus (264) Google Scholar).The origin-binding protein (UL9 protein) is a 94-kDa protein that recognizes specific elements in the HSV-1 origins of DNA replication (4Elias P. Lehman I.R. Proc. Natl. Acad. Sci. U. S. A. 1988; 85: 2959-2963Crossref PubMed Scopus (91) Google Scholar, 5Olivo P.D. Nelson N.J. Challberg M.D. Proc. Natl. Acad. Sci. U. S. A. 1988; 85: 5414-5418Crossref PubMed Scopus (121) Google Scholar). The UL9 protein also possesses intrinsic DNA helicase activity, and associated nucleoside triphosphatase (ATPase) activity, that is presumably required for unwinding the origins of DNA replication (6Bruckner R.C. Crute J.J. Dodson M.S. Lehman I.R. J. Biol. Chem. 1991; 266: 2669-2674Abstract Full Text PDF PubMed Google Scholar, 7Fierer D.S. Challberg M.D. J. Virol. 1992; 66: 3986-3995Crossref PubMed Google Scholar, 8Dodson M.S. Lehman I.R. J. Biol. Chem. 1993; 268: 1213-1219Abstract Full Text PDF PubMed Google Scholar, 9Boehmer P.E. Dodson M.S. Lehman I.R. J. Biol. Chem. 1993; 268: 1220-1225Abstract Full Text PDF PubMed Google Scholar). The HSV-1 SSB, henceforth referred to as ICP8 (infected cell polypeptide8), is a 128-kDa protein, capable of binding single-stranded DNA (ssDNA) cooperatively and with high affinity (10Ruyechan W.T. J. Virol. 1983; 46: 661-666Crossref PubMed Google Scholar). ICP8 forms a specific complex with the UL9 protein by interacting with its extreme C terminus (11Boehmer P.E. Lehman I.R. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 8444-8448Crossref PubMed Scopus (90) Google Scholar, 12Boehmer P.E. Craigie M.C. Stow N.D. Lehman I.R. J. Biol. Chem. 1994; 269: 29329-29334Abstract Full Text PDF PubMed Google Scholar, 13Gustafsson C.M. Falkenberg M. Simonsson S. Valadi H. Elias P. J. Biol. Chem. 1995; 270: 19028-19034Abstract Full Text Full Text PDF PubMed Scopus (23) Google Scholar). In addition, ICP8 has been shown to interact with the HSV-1 DNA polymerase and helicase-primase (14Littler D. Purifoy D. Minson A. Powell K.L. J. Gen. Virol. 1983; 64: 983-995Crossref PubMed Scopus (35) Google Scholar, 15Vaughan P.J. Banks L.M. Purifoy D.J. Powell K.L. J. Gen. Virol. 1984; 65: 2033-2041Crossref PubMed Scopus (40) Google Scholar, 16Chiou H.C. Weller S.K. Coen D.M. Virology. 1985; 145: 213-226Crossref PubMed Scopus (40) Google Scholar, 17Le Gac N.T. Villani G. Hoffman J.-S. Boehmer P.E. J. Biol. Chem. 1996; 271: 21645-21651Abstract Full Text Full Text PDF PubMed Scopus (51) Google Scholar, 18Falkenberg M. Bushnell D.A. Elias P. Lehman I.R. J. Biol. Chem. 1997; 272: 22766-22770Crossref PubMed Scopus (53) Google Scholar). The ability of ICP8 to participate in multiple protein-protein interactions suggests that it fulfills several roles during viral DNA replication.The interaction between ICP8 and UL9 protein greatly stimulates the rate and extent of DNA unwinding catalyzed by the UL9 protein, enabling it to unwind long stretches of DNA (9Boehmer P.E. Dodson M.S. Lehman I.R. J. Biol. Chem. 1993; 268: 1220-1225Abstract Full Text PDF PubMed Google Scholar, 19Makhov A.M. Boehmer P.E. Lehman I.R. Griffith J.D. J. Mol. Biol. 1996; 258: 789-799Crossref PubMed Scopus (39) Google Scholar). It has been shown that disruption of the ICP8-UL9 protein complex by deletion of the 27 C-terminal amino acid of the UL9 protein greatly reduces origin-specific DNA replication (12Boehmer P.E. Craigie M.C. Stow N.D. Lehman I.R. J. Biol. Chem. 1994; 269: 29329-29334Abstract Full Text PDF PubMed Google Scholar). Presumably, the ICP8-UL9 protein complex promotes efficient unwinding of the HSV-1 origins of DNA replication (20Makhov A.M. Boehmer P.E. Lehman I.R. Griffith J.D. EMBO J. 1996; 15: 1742-1750Crossref PubMed Scopus (46) Google Scholar, 21Lee S.S.-K. Lehman I.R. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 2838-2842Crossref PubMed Scopus (51) Google Scholar).This study examines the mechanism by which ICP8 stimulates the DNA helicase and ATPase activities of the UL9 protein. The results show that ICP8 increases the processivity of the UL9 protein, facilitating its translocation along DNA and through regions of secondary structure.DISCUSSIONThe aim of this study was to identify the mechanism by which the HSV-1 SSB, ICP8, stimulates the DNA helicase and DNA-dependent ATPase activities of the HSV-1 origin-binding protein (UL9 protein).The results show that subsaturating concentrations of ICP8 were sufficient to stimulate the DNA helicase activity of the UL9 protein, and that maximal stimulation occurred at an ICP8 to UL9 protein ratio of 1:1. This observation is consistent with the existence of a specific ICP8-UL9 protein complex that translocates along the DNA and is active during DNA unwinding. In addition, the stoichiometry of 1:1 inferred from the functional interaction between ICP8 and UL9 protein is identical to that observed for the physical complex (13Gustafsson C.M. Falkenberg M. Simonsson S. Valadi H. Elias P. J. Biol. Chem. 1995; 270: 19028-19034Abstract Full Text Full Text PDF PubMed Scopus (23) Google Scholar).ICP8 also stimulated the DNA-dependent ATPase activity of the UL9 protein in a species-specific manner. A heterologous SSB, E-SSB, led to inhibition of activity, presumably by preventing access of the UL9 protein to the DNA substrate. Furthermore, ICP8 partially reversed the inhibitory effect of E-SSB. These observations are indicative of a specific tertiary complex that consists of UL9 protein, ICP8, and ssDNA.Interestingly, the stimulatory effect of ICP8 on the DNA helicase and DNA-dependent ATPase activities of the UL9 protein was dependent on the ICP8/nucleotide ratio. Maximal stimulation was observed at subsaturating concentrations of ICP8, whereas coating concentrations produced less of an effect. Assuming that the level of stimulation correlates with the physical association of ICP8 and UL9 protein, these results suggest that the ICP8-UL9 protein complex is less stable at high ICP8/nucleotide ratios. This finding may explain why Gustafsson et al. (13Gustafsson C.M. Falkenberg M. Simonsson S. Valadi H. Elias P. J. Biol. Chem. 1995; 270: 19028-19034Abstract Full Text Full Text PDF PubMed Scopus (23) Google Scholar) failed to detect a physical complex of UL9 protein, ICP8, and ssDNA at high ICP8/nucleotide ratios. It is possible that, when ICP8 is in excess and all ssDNA sites are occupied, ICP8 undergoes a conformational change and loses its affinity for UL9 protein.The existence of a lag in the initial phase of the DNA unwinding reaction catalyzed by the UL9 protein suggests that the rate-limiting step is its association with the DNA substrate. This conclusion is substantiated by the observation that the lag was eliminated by preincubating the UL9 protein and DNA substrate. Moreover, rapid and maximal stimulation of DNA unwinding by ICP8 was only observed when UL9 protein was preincubated with DNA substrate or with ongoing DNA helicase reactions. Consequently, ICP8 exerts its maximal effect on a preassembled UL9 protein-DNA complex.The observation that ICP8 had no effect on the DNA-independent ATPase activity of the UL9 protein suggests that it does not directly affect the catalytic site of the UL9 protein. Therefore, experiments were designed to examine how ICP8 influences the interaction of the UL9 protein with its DNA substrate during DNA unwinding and DNA-dependent ATP hydrolysis. Both activities entail translocation of the UL9 protein along ssDNA and through regions of duplex DNA. Consequently, these experiments addressed how ICP8 affects the translocation of UL9 protein along DNA.Addition of excess challenger DNA to ongoing DNA unwinding reactions showed that UL9 protein was effectively competed from the DNA substrate, implying that it dissociates rapidly from the DNA substrate and that it is nonprocessive. The inhibitory effect of the competitor DNA was eliminated by ICP8. Specifically, equimolar, subsaturating concentrations of ICP8 added immediately prior to competitor DNA prevented the effect of the challenger DNA. These results suggest that ICP8 prevents the dissociation of UL9 protein from the DNA substrate and therefore increases its processivity. Further evidence for the processivity enhancing function of ICP8 is provided by previous experiments in which ICP8 enabled the UL9 protein to unwind long regions of DNA (up to ∼3 kilobase pairs) (9Boehmer P.E. Dodson M.S. Lehman I.R. J. Biol. Chem. 1993; 268: 1220-1225Abstract Full Text PDF PubMed Google Scholar, 19Makhov A.M. Boehmer P.E. Lehman I.R. Griffith J.D. J. Mol. Biol. 1996; 258: 789-799Crossref PubMed Scopus (39) Google Scholar).(dT)15 is sufficient to elicit the DNA-dependent ATPase activity of the UL9 protein (8Dodson M.S. Lehman I.R. J. Biol. Chem. 1993; 268: 1213-1219Abstract Full Text PDF PubMed Google Scholar). Presumably, this cofactor is sufficiently long to allow the UL9 protein to bind but is too short to allow translocation of the UL9 protein along the DNA. The extent of ATP hydrolysis seen with this cofactor should therefore be representative of UL9 protein binding only. Consistent with the assumption that ICP8 increases the processivity of the UL9 protein, there was no effect of ICP8 on the rate of ATP hydrolysis with (dT)15. In contrast, ATP hydrolysis with longer DNA cofactors, which allow both binding and translocation of the UL9 protein, was greatly stimulated by ICP8. Furthermore, ICP8 had an even greater stimulatory effect on ATP hydrolysis with DNA cofactors that contain secondary structure.In conclusion, the ability of ICP8 to prevent competition with challenger DNA during DNA unwinding, and to stimulate ATP hydrolysis with DNA cofactors that allow translocation, implies that it prevents dissociation of the UL9 protein from the DNA substrate and increases its processivity. In addition, ICP8 appears to facilitate translocation of the UL9 protein through regions of duplex DNA. This is probably a manifestation of the helix-destabilizing activity of ICP8 (24Boehmer P.E. Lehman I.R. J. Virol. 1993; 67: 711-715Crossref PubMed Google Scholar).Fig. 9 shows a model of how ICP8 stimulates the translocation of the UL9 protein along ssDNA and through regions of duplex DNA. Both molecules within the UL9 protein dimer (6Bruckner R.C. Crute J.J. Dodson M.S. Lehman I.R. J. Biol. Chem. 1991; 266: 2669-2674Abstract Full Text PDF PubMed Google Scholar,7Fierer D.S. Challberg M.D. J. Virol. 1992; 66: 3986-3995Crossref PubMed Google Scholar, 19Makhov A.M. Boehmer P.E. Lehman I.R. Griffith J.D. J. Mol. Biol. 1996; 258: 789-799Crossref PubMed Scopus (39) Google Scholar, 20Makhov A.M. Boehmer P.E. Lehman I.R. Griffith J.D. EMBO J. 1996; 15: 1742-1750Crossref PubMed Scopus (46) Google Scholar) make contact with ssDNA via their N-terminal ssDNA-binding regions (25Abbotts A.P. Stow N.D. J. Gen. Virol. 1995; 76: 3125-3130Crossref PubMed Scopus (18) Google Scholar), enabling it to translocate 3′ to 5′ (7Fierer D.S. Challberg M.D. J. Virol. 1992; 66: 3986-3995Crossref PubMed Google Scholar, 9Boehmer P.E. Dodson M.S. Lehman I.R. J. Biol. Chem. 1993; 268: 1220-1225Abstract Full Text PDF PubMed Google Scholar). One molecule of ICP8 is bound to the C terminus of each UL9 protein molecule (11Boehmer P.E. Lehman I.R. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 8444-8448Crossref PubMed Scopus (90) Google Scholar,12Boehmer P.E. Craigie M.C. Stow N.D. Lehman I.R. J. Biol. Chem. 1994; 269: 29329-29334Abstract Full Text PDF PubMed Google Scholar), allowing one ICP8 molecule to bind the template strand, tethering the UL9 protein to the DNA substrate, while the second molecule of ICP8 assimilates and stabilizes the unwound primer strand, thereby facilitating the translocation of the UL9 protein through regions of duplex DNA.Previous studies have shown that a site-specific cisplatin lesion impairs the DNA helicase activity of the UL9 protein (22Villani G. Pillaire M.J. Boehmer P.E. J. Biol. Chem. 1994; 269: 21676-21681Abstract Full Text PDF PubMed Google Scholar). Furthermore, it was shown that ICP8 relieved the inhibitory effect imposed by the lesion. Based on the findings in this study, it is likely that ICP8 enables the UL9 protein to bypass the lesion by tethering it to the DNA substrate, thereby preventing its dissociation.The effects of cognate and noncognate SSBs have been documented for numerous DNA helicases (reviewed in Refs. 26Kornberg A. Baker T. DNA Replication. W. H. Freeman & Co., New York1992Google Scholar, 27Hübscher U. Maga G. Podust V.N. DePamphilis M.J. DNA Replication in Eukaryotic Cells. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY1996: 525-543Google Scholar, 28Borowiec J.A. DePamphilis M.J. DNA Replication in Eukaryotic cells. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY1996: 545-574Google Scholar). E-SSB has been shown to affect the activities of E. coli PriA (29Lee M.S. Marians K.J. Proc. Natl. Acad. Sci. U. S. A. 1987; 84: 8345-8349Crossref PubMed Scopus (83) Google Scholar, 30Lasken R.S. Kornberg A. J. Biol. Chem. 1988; 263: 5512-5518Abstract Full Text PDF PubMed Google Scholar), DNA helicase IV (31Yancey-Wrona J.E. Wood E.R. George J.W. Smith K.R. Matson S.W. Eur. J. Biochem. 1992; 207: 479-485Crossref PubMed Scopus (15) Google Scholar), and Rep (32Scott J.F. Eisenberg S. Bertsch L. Kornberg A. Proc. Natl. Acad. Sci. U. S. A. 1977; 74: 193-197Crossref PubMed Scopus (134) Google Scholar, 33Yarranton G.T. Gefter M.L. Proc. Natl. Acad. Sci. U. S. A. 1979; 76: 1658-1662Crossref PubMed Scopus (149) Google Scholar) DNA helicases. Likewise, replication protein-A (RP-A) has been shown to affect several eukaryotic DNA helicases including: Saccharomyces cerevisiae Hel B (HCSB) (34Biswas E.E. Chen P.H. Biswas S.B. Biochem. 1993; 32: 13393-13398Crossref PubMed Scopus (17) Google Scholar, 35Biswas E.E. Chen P.H. Leszyk J. Biswas S.B. Biochem. Biophys. Res. Commun. 1995; 206: 850-856Crossref PubMed Scopus (19) Google Scholar), calf thymus DNA helicases A–D and F (36Thömmes P. Ferrari E. Jessberger R. Hübscher U. J. Biol. Chem. 1992; 267: 6063-6073Abstract Full Text PDF PubMed Google Scholar, 37Zhang S. Grosse F. FEBS Lett. 1992; 312: 143-146Crossref PubMed Scopus (9) Google Scholar, 38Georgaki A. Tuteja N. Sturzenegger B. Hübscher U. Nucleic Acids Res. 1994; 22: 1128-1134Crossref PubMed Scopus (17) Google Scholar), human DNA helicases isolated from HeLa cells (39Seo Y.-S. Lee S.H. Hurwitz J. J. Biol. Chem. 1991; 266: 13161-13170Abstract Full Text PDF PubMed Google Scholar, 40Seo Y.-S. Hurwitz J. J. Biol. Chem. 1993; 268: 10282-10295Abstract Full Text PDF PubMed Google Scholar), and simian virus 40 large T antigen (41Dodson M. Dean F. Bullock P. Echols H. Hurwitz J. Science. 1987; 238: 964-967Crossref PubMed Scopus (98) Google Scholar, 42Wold M. Li J. Kelly T. Proc. Natl. Acad. Sci. U. S. A. 1987; 84: 3643-3647Crossref PubMed Scopus (159) Google Scholar, 43Dornreiter I. Erdile L.F. Gilbert I.U. von Winkler D. Kelly T.J. Fanning E. EMBO J. 1992; 11: 769-776Crossref PubMed Scopus (284) Google Scholar). In HSV-1, apart from the interaction between ICP8 and the UL9 protein discussed in this report, it has also been shown that ICP8 can specifically stimulate the DNA helicase activity of the DNA helicase-primase (17Le Gac N.T. Villani G. Hoffman J.-S. Boehmer P.E. J. Biol. Chem. 1996; 271: 21645-21651Abstract Full Text Full Text PDF PubMed Scopus (51) Google Scholar, 18Falkenberg M. Bushnell D.A. Elias P. Lehman I.R. J. Biol. Chem. 1997; 272: 22766-22770Crossref PubMed Scopus (53) Google Scholar, 44Crute J.J. Lehman I.R. J. Biol. Chem. 1991; 266: 4484-4488Abstract Full Text PDF PubMed Google Scholar). In most cases, SSBs have been shown to enhance DNA unwinding by preventing nonproductive binding of the DNA helicase to the DNA substrate. Specific protein-protein interactions between DNA helicases and cognate SSBs have also been shown to increase the length of DNA unwound. However, no previous studies have addressed the exact mechanism by which an SSB stimulates DNA unwinding.Previous studies have indicated the importance of the ICP8-UL9 protein interaction for HSV-1 origin-specific DNA replication (12Boehmer P.E. Craigie M.C. Stow N.D. Lehman I.R. J. Biol. Chem. 1994; 269: 29329-29334Abstract Full Text PDF PubMed Google Scholar, 21Lee S.S.-K. Lehman I.R. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 2838-2842Crossref PubMed Scopus (51) Google Scholar). The properties of the UL9 protein and ICP8 and those of the ICP8-UL9 protein complex, described in this and previous studies (7Fierer D.S. Challberg M.D. J. Virol. 1992; 66: 3986-3995Crossref PubMed Google Scholar, 9Boehmer P.E. Dodson M.S. Lehman I.R. J. Biol. Chem. 1993; 268: 1220-1225Abstract Full Text PDF PubMed Google Scholar, 10Ruyechan W.T. J. Virol. 1983; 46: 661-666Crossref PubMed Google Scholar, 20Makhov A.M. Boehmer P.E. Lehman I.R. Griffith J.D. EMBO J. 1996; 15: 1742-1750Crossref PubMed Scopus (46) Google Scholar,21Lee S.S.-K. Lehman I.R. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 2838-2842Crossref PubMed Scopus (51) Google Scholar, 24Boehmer P.E. Lehman I.R. J. Virol. 1993; 67: 711-715Crossref PubMed Google Scholar), illustrate how this complex is suited for its predicted role of recognizing and unwinding the HSV-1 origins of DNA replication. Herpes simplex virus type-1 (HSV-1) 1The abbreviations used are: HSV-1, herpes simplex virus type-1; SSB, single-strand DNA-binding protein; ssDNA, single-stranded DNA; E-SSB, E. coli SSB; EPPS, N-(2-hydroxyethyl)piperazine-N′-(3-propanesulfonic acid). 1The abbreviations used are: HSV-1, herpes simplex virus type-1; SSB, single-strand DNA-binding protein; ssDNA, single-stranded DNA; E-SSB, E. coli SSB; EPPS, N-(2-hydroxyethyl)piperazine-N′-(3-propanesulfonic acid). is a double-stranded DNA virus with a genome of ∼152 kilobase pairs that contains three origins of DNA replication (1Boehmer P.E. Lehman I.R. Annu. Rev. Biochem. 1997; 66: 347-384Crossref PubMed Scopus (264) Google Scholar). Replication of origin-containing plasmids requires the action of seven viral gene products (2Wu C.A. Nelson N.J. McGeoch D.J. Challberg M.D. J. Virol. 1988; 62: 435-443Crossref PubMed Google Scholar, 3McGeoch D.J. Dalrymple M.A. Dolan A. McNab D. Perry L.J. Taylor P. Challberg M.D. J. Virol. 1988; 62: 444-453Crossref PubMed Google Scholar). These seven gene products comprise a highly processive heterodimeric DNA polymerase (UL30/UL42 genes), a heterotrimeric DNA helicase-primase (UL5/UL8/UL52genes), a single-strand DNA-binding protein (SSB) (UL29gene), and an origin-binding protein (UL9 gene) (reviewed in Ref. 1Boehmer P.E. Lehman I.R. Annu. Rev. Biochem. 1997; 66: 347-384Crossref PubMed Scopus (264) Google Scholar). The origin-binding protein (UL9 protein) is a 94-kDa protein that recognizes specific elements in the HSV-1 origins of DNA replication (4Elias P. Lehman I.R. Proc. Natl. Acad. Sci. U. S. A. 1988; 85: 2959-2963Crossref PubMed Scopus (91) Google Scholar, 5Olivo P.D. Nelson N.J. Challberg M.D. Proc. Natl. Acad. Sci. U. S. A. 1988; 85: 5414-5418Crossref PubMed Scopus (121) Google Scholar). The UL9 protein also possesses intrinsic DNA helicase activity, and associated nucleoside triphosphatase (ATPase) activity, that is presumably required for unwinding the origins of DNA replication (6Bruckner R.C. Crute J.J. Dodson M.S. Lehman I.R. J. Biol. Chem. 1991; 266: 2669-2674Abstract Full Text PDF PubMed Google Scholar, 7Fierer D.S. Challberg M.D. J. Virol. 1992; 66: 3986-3995Crossref PubMed Google Scholar, 8Dodson M.S. Lehman I.R. J. Biol. Chem. 1993; 268: 1213-1219Abstract Full Text PDF PubMed Google Scholar, 9Boehmer P.E. Dodson M.S. Lehman I.R. J. Biol. Chem. 1993; 268: 1220-1225Abstract Full Text PDF PubMed Google Scholar). The HSV-1 SSB, henceforth referred to as ICP8 (infected cell polypeptide8), is a 128-kDa protein, capable of binding single-stranded DNA (ssDNA) cooperatively and with high affinity (10Ruyechan W.T. J. Virol. 1983; 46: 661-666Crossref PubMed Google Scholar). ICP8 forms a specific complex with the UL9 protein by interacting with its extreme C terminus (11Boehmer P.E. Lehman I.R. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 8444-8448Crossref PubMed Scopus (90) Google Scholar, 12Boehmer P.E. Craigie M.C. Stow N.D. Lehman I.R. J. Biol. Chem. 1994; 269: 29329-29334Abstract Full Text PDF PubMed Google Scholar, 13Gustafsson C.M. Falkenberg M. Simonsson S. Valadi H. Elias P. J. Biol. Chem. 1995; 270: 19028-19034Abstract Full Text Full Text PDF PubMed Scopus (23) Google Scholar). In addition, ICP8 has been shown to interact with the HSV-1 DNA polymerase and helicase-primase (14Littler D. Purifoy D. Minson A. Powell K.L. J. Gen. Virol. 1983; 64: 983-995Crossref PubMed Scopus (35) Google Scholar, 15Vaughan P.J. Banks L.M. Purifoy D.J. Powell K.L. J. Gen. Virol. 1984; 65: 2033-2041Crossref PubMed Scopus (40) Google Scholar, 16Chiou H.C. Weller S.K. Coen D.M. Virology. 1985; 145: 213-226Crossref PubMed Scopus (40) Google Scholar, 17Le Gac N.T. Villani G. Hoffman J.-S. Boehmer P.E. J. Biol. Chem. 1996; 271: 21645-21651Abstract Full Text Full Text PDF PubMed Scopus (51) Google Scholar, 18Falkenberg M. Bushnell D.A. Elias P. Lehman I.R. J. Biol. Chem. 1997; 272: 22766-22770Crossref PubMed Scopus (53) Google Scholar). The ability of ICP8 to participate in multiple protein-protein interactions suggests that it fulfills several roles during viral DNA replication. The interaction between ICP8 and UL9 protein greatly stimulates the rate and extent of DNA unwinding catalyzed by the UL9 protein, enabling it to unwind long stretches of DNA (9Boehmer P.E. Dodson M.S. Lehman I.R. J. Biol. Chem. 1993; 268: 1220-1225Abstract Full Text PDF PubMed Google Scholar, 19Makhov A.M. Boehmer P.E. Lehman I.R. Griffith J.D. J. Mol. Biol. 1996; 258: 789-799Crossref PubMed Scopus (39) Google Scholar). It has been shown that disruption of the ICP8-UL9 protein complex by deletion of the 27 C-terminal amino acid of the UL9 protein greatly reduces origin-specific DNA replication (12Boehmer P.E. Craigie M.C. Stow N.D. Lehman I.R. J. Biol. Chem. 1994; 269: 29329-29334Abstract Full Text PDF PubMed Google Scholar). Presumably, the ICP8-UL9 protein complex promotes efficient unwinding of the HSV-1 origins of DNA replication (20Makhov A.M. Boehmer P.E. Lehman I.R. Griffith J.D. EMBO J. 1996; 15: 1742-1750Crossref PubMed Scopus (46) Google Scholar, 21Lee S.S.-K. Lehman I.R. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 2838-2842Crossref PubMed Scopus (51) Google Scholar). This study examines the mechanism by which ICP8 stimulates the DNA helicase and ATPase activities of the UL9 protein. The results show that ICP8 increases the processivity of the UL9 protein, facilitating its translocation along DNA and through regions of secondary structure. DISCUSSIONThe aim of this study was to identify the mechanism by which the HSV-1 SSB, ICP8, stimulates the DNA helicase and DNA-dependent ATPase activities of the HSV-1 origin-binding protein (UL9 protein).The results show that subsaturating concentrations of ICP8 were sufficient to stimulate the DNA helicase activity of the UL9 protein, and that maximal stimulation occurred at an ICP8 to UL9 protein ratio of 1:1. This observation is consistent with the existence of a specific ICP8-UL9 protein complex that translocates along the DNA and is active during DNA unwinding. In addition, the stoichiometry of 1:1 inferred from the functional interaction between ICP8 and UL9 protein is identical to that observed for the physical complex (13Gustafsson C.M. Falkenberg M. Simonsson S. Valadi H. Elias P. J. Biol. Chem. 1995; 270: 19028-19034Abstract Full Text Full Text PDF PubMed Scopus (23) Google Scholar).ICP8 also stimulated the DNA-dependent ATPase activity of the UL9 protein in a species-specific manner. A heterologous SSB, E-SSB, led to inhibition of activity, presumably by preventing access of the UL9 protein to the DNA substrate. Furthermore, ICP8 partially reversed the inhibitory effect of E-SSB. These observations are indicative of a specific tertiary complex that consists of UL9 protein, ICP8, and ssDNA.Interestingly, the stimulatory effect of ICP8 on the DNA helicase and DNA-dependent ATPase activities of the UL9 protein was dependent on the ICP8/nucleotide ratio. Maximal stimulation was observed at subsaturating concentrations of ICP8, whereas coating concentrations produced less of an effect. Assuming that the level of stimulation correlates with the physical association of ICP8 and UL9 protein, these results suggest that the ICP8-UL9 protein complex is less stable at high ICP8/nucleotide ratios. This finding may explain why Gustafsson et al. (13Gustafsson C.M. Falkenberg M. Simonsson S. Valadi H. Elias P. J. Biol. Chem. 1995; 270: 19028-19034Abstract Full Text Full Text PDF PubMed Scopus (23) Google Scholar) failed to detect a physical complex of UL9 protein, ICP8, and ssDNA at high ICP8/nucleotide ratios. It is possible that, when ICP8 is in excess and all ssDNA sites are occupied, ICP8 undergoes a conformational change and loses its affinity for UL9 protein.The existence of a lag in the initial phase of the DNA unwinding reaction catalyzed by the UL9 protein suggests that the rate-limiting step is its association with the DNA substrate. This conclusion is substantiated by the observation that the lag was eliminated by preincubating the UL9 protein and DNA substrate. Moreover, rapid and maximal stimulation of DNA unwinding by ICP8 was only observed when UL9 protein was preincubated with DNA substrate or with ongoing DNA helicase reactions. Consequently, ICP8 exerts its maximal effect on a preassembled UL9 protein-DNA complex.The observation that ICP8 had no effect on the DNA-independent ATPase activity of the UL9 protein suggests that it does not directly affect the catalytic site of the UL9 protein. Therefore, experiments were designed to examine how ICP8 influences the interaction of the UL9 protein with its DNA substrate during DNA unwinding and DNA-dependent ATP hydrolysis. Both activities entail translocation of the UL9 protein along ssDNA and through regions of duplex DNA. Consequently, these experiments addressed how ICP8 affects the translocation of UL9 protein along DNA.Addition of excess challenger DNA to ongoing DNA unwinding reactions showed that UL9 protein was effectively competed from the DNA substrate, implying that it dissociates rapidly from the DNA substrate and that it is nonprocessive. The inhibitory effect of the competitor DNA was eliminated by ICP8. Specifically, equimolar, subsaturating concentrations of ICP8 added immediately prior to competitor DNA prevented the effect of the challenger DNA. These results suggest that ICP8 prevents the dissociation of UL9 protein from the DNA substrate and therefore increases its processivity. Further evidence for the processivity enhancing function of ICP8 is provided by previous experiments in which ICP8 enabled the UL9 protein to unwind long regions of DNA (up to ∼3 kilobase pairs) (9Boehmer P.E. Dodson M.S. Lehman I.R. J. Biol. Chem. 1993; 268: 1220-1225Abstract Full Text PDF PubMed Google Scholar, 19Makhov A.M. Boehmer P.E. Lehman I.R. Griffith J.D. J. Mol. Biol. 1996; 258: 789-799Crossref PubMed Scopus (39) Google Scholar).(dT)15 is sufficient to elicit the DNA-dependent ATPase activity of the UL9 protein (8Dodson M.S. Lehman I.R. J. Biol. Chem. 1993; 268: 1213-1219Abstract Full Text PDF PubMed Google Scholar). Presumably, this cofactor is sufficiently long to allow the UL9 protein to bind but is too short to allow translocation of the UL9 protein along the DNA. The extent of ATP hydrolysis seen with this cofactor should therefore be representative of UL9 protein binding only. Consistent with the assumption that ICP8 increases the processivity of the UL9 protein, there was no effect of ICP8 on the rate of ATP hydrolysis with (dT)15. In contrast, ATP hydrolysis with longer DNA cofactors, which allow both binding and translocation of the UL9 protein, was greatly stimulated by ICP8. Furthermore, ICP8 had an even greater stimulatory effect on ATP hydrolysis with DNA cofactors that contain secondary structure.In conclusion, the ability of ICP8 to prevent competition with challenger DNA during DNA unwinding, and to stimulate ATP hydrolysis with DNA cofactors that allow translocation, implies that it prevents dissociation of the UL9 protein from the DNA substrate and increases its processivity. In addition, ICP8 appears to facilitate translocation of the UL9 protein through regions of duplex DNA. This is probably a manifestation of the helix-destabilizing activity of ICP8 (24Boehmer P.E. Lehman I.R. J. Virol. 1993; 67: 711-715Crossref PubMed Google Scholar).Fig. 9 shows a model of how ICP8 stimulates the translocation of the UL9 protein along ssDNA and through regions of duplex DNA. Both molecules within the UL9 protein dimer (6Bruckner R.C. Crute J.J. Dodson M.S. Lehman I.R. J. Biol. Chem. 1991; 266: 2669-2674Abstract Full Text PDF PubMed Google Scholar,7Fierer D.S. Challberg M.D. J. Virol. 1992; 66: 3986-3995Crossref PubMed Google Scholar, 19Makhov A.M. Boehmer P.E. Lehman I.R. Griffith J.D. J. Mol. Biol. 1996; 258: 789-799Crossref PubMed Scopus (39) Google Scholar, 20Makhov A.M. Boehmer P.E. Lehman I.R. Griffith J.D. EMBO J. 1996; 15: 1742-1750Crossref PubMed Scopus (46) Google Scholar) make contact with ssDNA via their N-terminal ssDNA-binding regions (25Abbotts A.P. Stow N.D. J. Gen. Virol. 1995; 76: 3125-3130Crossref PubMed Scopus (18) Google Scholar), enabling it to translocate 3′ to 5′ (7Fierer D.S. Challberg M.D. J. Virol. 1992; 66: 3986-3995Crossref PubMed Google Scholar, 9Boehmer P.E. Dodson M.S. Lehman I.R. J. Biol. Chem. 1993; 268: 1220-1225Abstract Full Text PDF PubMed Google Scholar). One molecule of ICP8 is bound to the C terminus of each UL9 protein molecule (11Boehmer P.E. Lehman I.R. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 8444-8448Crossref PubMed Scopus (90) Google Scholar,12Boehmer P.E. Craigie M.C. Stow N.D. Lehman I.R. J. Biol. Chem. 1994; 269: 29329-29334Abstract Full Text PDF PubMed Google Scholar), allowing one ICP8 molecule to bind the template strand, tethering the UL9 protein to the DNA substrate, while the second molecule of ICP8 assimilates and stabilizes the unwound primer strand, thereby facilitating the translocation of the UL9 protein through regions of duplex DNA.Previous studies have shown that a site-specific cisplatin lesion impairs the DNA helicase activity of the UL9 protein (22Villani G. Pillaire M.J. Boehmer P.E. J. Biol. Chem. 1994; 269: 21676-21681Abstract Full Text PDF PubMed Google Scholar). Furthermore, it was shown that ICP8 relieved the inhibitory effect imposed by the lesion. Based on the findings in this study, it is likely that ICP8 enables the UL9 protein to bypass the lesion by tethering it to the DNA substrate, thereby preventing its dissociation.The effects of cognate and noncognate SSBs have been documented for numerous DNA helicases (reviewed in Refs. 26Kornberg A. Baker T. DNA Replication. W. H. Freeman & Co., New York1992Google Scholar, 27Hübscher U. Maga G. Podust V.N. DePamphilis M.J. DNA Replication in Eukaryotic Cells. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY1996: 525-543Google Scholar, 28Borowiec J.A. DePamphilis M.J. DNA Replication in Eukaryotic cells. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY1996: 545-574Google Scholar). E-SSB has been shown to affect the activities of E. coli PriA (29Lee M.S. Marians K.J. Proc. Natl. Acad. Sci. U. S. A. 1987; 84: 8345-8349Crossref PubMed Scopus (83) Google Scholar, 30Lasken R.S. Kornberg A. J. Biol. Chem. 1988; 263: 5512-5518Abstract Full Text PDF PubMed Google Scholar), DNA helicase IV (31Yancey-Wrona J.E. Wood E.R. George J.W. Smith K.R. Matson S.W. Eur. J. Biochem. 1992; 207: 479-485Crossref PubMed Scopus (15) Google Scholar), and Rep (32Scott J.F. Eisenberg S. Bertsch L. Kornberg A. Proc. Natl. Acad. Sci. U. S. A. 1977; 74: 193-197Crossref PubMed Scopus (134) Google Scholar, 33Yarranton G.T. Gefter M.L. Proc. Natl. Acad. Sci. U. S. A. 1979; 76: 1658-1662Crossref PubMed Scopus (149) Google Scholar) DNA helicases. Likewise, replication protein-A (RP-A) has been shown to affect several eukaryotic DNA helicases including: Saccharomyces cerevisiae Hel B (HCSB) (34Biswas E.E. Chen P.H. Biswas S.B. Biochem. 1993; 32: 13393-13398Crossref PubMed Scopus (17) Google Scholar, 35Biswas E.E. Chen P.H. Leszyk J. Biswas S.B. Biochem. Biophys. Res. Commun. 1995; 206: 850-856Crossref PubMed Scopus (19) Google Scholar), calf thymus DNA helicases A–D and F (36Thömmes P. Ferrari E. Jessberger R. Hübscher U. J. Biol. Chem. 1992; 267: 6063-6073Abstract Full Text PDF PubMed Google Scholar, 37Zhang S. Grosse F. FEBS Lett. 1992; 312: 143-146Crossref PubMed Scopus (9) Google Scholar, 38Georgaki A. Tuteja N. Sturzenegger B. Hübscher U. Nucleic Acids Res. 1994; 22: 1128-1134Crossref PubMed Scopus (17) Google Scholar), human DNA helicases isolated from HeLa cells (39Seo Y.-S. Lee S.H. Hurwitz J. J. Biol. Chem. 1991; 266: 13161-13170Abstract Full Text PDF PubMed Google Scholar, 40Seo Y.-S. Hurwitz J. J. Biol. Chem. 1993; 268: 10282-10295Abstract Full Text PDF PubMed Google Scholar), and simian virus 40 large T antigen (41Dodson M. Dean F. Bullock P. Echols H. Hurwitz J. Science. 1987; 238: 964-967Crossref PubMed Scopus (98) Google Scholar, 42Wold M. Li J. Kelly T. Proc. Natl. Acad. Sci. U. S. A. 1987; 84: 3643-3647Crossref PubMed Scopus (159) Google Scholar, 43Dornreiter I. Erdile L.F. Gilbert I.U. von Winkler D. Kelly T.J. Fanning E. EMBO J. 1992; 11: 769-776Crossref PubMed Scopus (284) Google Scholar). In HSV-1, apart from the interaction between ICP8 and the UL9 protein discussed in this report, it has also been shown that ICP8 can specifically stimulate the DNA helicase activity of the DNA helicase-primase (17Le Gac N.T. Villani G. Hoffman J.-S. Boehmer P.E. J. Biol. Chem. 1996; 271: 21645-21651Abstract Full Text Full Text PDF PubMed Scopus (51) Google Scholar, 18Falkenberg M. Bushnell D.A. Elias P. Lehman I.R. J. Biol. Chem. 1997; 272: 22766-22770Crossref PubMed Scopus (53) Google Scholar, 44Crute J.J. Lehman I.R. J. Biol. Chem. 1991; 266: 4484-4488Abstract Full Text PDF PubMed Google Scholar). In most cases, SSBs have been shown to enhance DNA unwinding by preventing nonproductive binding of the DNA helicase to the DNA substrate. Specific protein-protein interactions between DNA helicases and cognate SSBs have also been shown to increase the length of DNA unwound. However, no previous studies have addressed the exact mechanism by which an SSB stimulates DNA unwinding.Previous studies have indicated the importance of the ICP8-UL9 protein interaction for HSV-1 origin-specific DNA replication (12Boehmer P.E. Craigie M.C. Stow N.D. Lehman I.R. J. Biol. Chem. 1994; 269: 29329-29334Abstract Full Text PDF PubMed Google Scholar, 21Lee S.S.-K. Lehman I.R. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 2838-2842Crossref PubMed Scopus (51) Google Scholar). The properties of the UL9 protein and ICP8 and those of the ICP8-UL9 protein complex, described in this and previous studies (7Fierer D.S. Challberg M.D. J. Virol. 1992; 66: 3986-3995Crossref PubMed Google Scholar, 9Boehmer P.E. Dodson M.S. Lehman I.R. J. Biol. Chem. 1993; 268: 1220-1225Abstract Full Text PDF PubMed Google Scholar, 10Ruyechan W.T. J. Virol. 1983; 46: 661-666Crossref PubMed Google Scholar, 20Makhov A.M. Boehmer P.E. Lehman I.R. Griffith J.D. EMBO J. 1996; 15: 1742-1750Crossref PubMed Scopus (46) Google Scholar,21Lee S.S.-K. Lehman I.R. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 2838-2842Crossref PubMed Scopus (51) Google Scholar, 24Boehmer P.E. Lehman I.R. J. Virol. 1993; 67: 711-715Crossref PubMed Google Scholar), illustrate how this complex is suited for its predicted role of recognizing and unwinding the HSV-1 origins of DNA replication. The aim of this study was to identify the mechanism by which the HSV-1 SSB, ICP8, stimulates the DNA helicase and DNA-dependent ATPase activities of the HSV-1 origin-binding protein (UL9 protein). The results show that subsaturating concentrations of ICP8 were sufficient to stimulate the DNA helicase activity of the UL9 protein, and that maximal stimulation occurred at an ICP8 to UL9 protein ratio of 1:1. This observation is consistent with the existence of a specific ICP8-UL9 protein complex that translocates along the DNA and is active during DNA unwinding. In addition, the stoichiometry of 1:1 inferred from the functional interaction between ICP8 and UL9 protein is identical to that observed for the physical complex (13Gustafsson C.M. Falkenberg M. Simonsson S. Valadi H. Elias P. J. Biol. Chem. 1995; 270: 19028-19034Abstract Full Text Full Text PDF PubMed Scopus (23) Google Scholar). ICP8 also stimulated the DNA-dependent ATPase activity of the UL9 protein in a species-specific manner. A heterologous SSB, E-SSB, led to inhibition of activity, presumably by preventing access of the UL9 protein to the DNA substrate. Furthermore, ICP8 partially reversed the inhibitory effect of E-SSB. These observations are indicative of a specific tertiary complex that consists of UL9 protein, ICP8, and ssDNA. Interestingly, the stimulatory effect of ICP8 on the DNA helicase and DNA-dependent ATPase activities of the UL9 protein was dependent on the ICP8/nucleotide ratio. Maximal stimulation was observed at subsaturating concentrations of ICP8, whereas coating concentrations produced less of an effect. Assuming that the level of stimulation correlates with the physical association of ICP8 and UL9 protein, these results suggest that the ICP8-UL9 protein complex is less stable at high ICP8/nucleotide ratios. This finding may explain why Gustafsson et al. (13Gustafsson C.M. Falkenberg M. Simonsson S. Valadi H. Elias P. J. Biol. Chem. 1995; 270: 19028-19034Abstract Full Text Full Text PDF PubMed Scopus (23) Google Scholar) failed to detect a physical complex of UL9 protein, ICP8, and ssDNA at high ICP8/nucleotide ratios. It is possible that, when ICP8 is in excess and all ssDNA sites are occupied, ICP8 undergoes a conformational change and loses its affinity for UL9 protein. The existence of a lag in the initial phase of the DNA unwinding reaction catalyzed by the UL9 protein suggests that the rate-limiting step is its association with the DNA substrate. This conclusion is substantiated by the observation that the lag was eliminated by preincubating the UL9 protein and DNA substrate. Moreover, rapid and maximal stimulation of DNA unwinding by ICP8 was only observed when UL9 protein was preincubated with DNA substrate or with ongoing DNA helicase reactions. Consequently, ICP8 exerts its maximal effect on a preassembled UL9 protein-DNA complex. The observation that ICP8 had no effect on the DNA-independent ATPase activity of the UL9 protein suggests that it does not directly affect the catalytic site of the UL9 protein. Therefore, experiments were designed to examine how ICP8 influences the interaction of the UL9 protein with its DNA substrate during DNA unwinding and DNA-dependent ATP hydrolysis. Both activities entail translocation of the UL9 protein along ssDNA and through regions of duplex DNA. Consequently, these experiments addressed how ICP8 affects the translocation of UL9 protein along DNA. Addition of excess challenger DNA to ongoing DNA unwinding reactions showed that UL9 protein was effectively competed from the DNA substrate, implying that it dissociates rapidly from the DNA substrate and that it is nonprocessive. The inhibitory effect of the competitor DNA was eliminated by ICP8. Specifically, equimolar, subsaturating concentrations of ICP8 added immediately prior to competitor DNA prevented the effect of the challenger DNA. These results suggest that ICP8 prevents the dissociation of UL9 protein from the DNA substrate and therefore increases its processivity. Further evidence for the processivity enhancing function of ICP8 is provided by previous experiments in which ICP8 enabled the UL9 protein to unwind long regions of DNA (up to ∼3 kilobase pairs) (9Boehmer P.E. Dodson M.S. Lehman I.R. J. Biol. Chem. 1993; 268: 1220-1225Abstract Full Text PDF PubMed Google Scholar, 19Makhov A.M. Boehmer P.E. Lehman I.R. Griffith J.D. J. Mol. Biol. 1996; 258: 789-799Crossref PubMed Scopus (39) Google Scholar). (dT)15 is sufficient to elicit the DNA-dependent ATPase activity of the UL9 protein (8Dodson M.S. Lehman I.R. J. Biol. Chem. 1993; 268: 1213-1219Abstract Full Text PDF PubMed Google Scholar). Presumably, this cofactor is sufficiently long to allow the UL9 protein to bind but is too short to allow translocation of the UL9 protein along the DNA. The extent of ATP hydrolysis seen with this cofactor should therefore be representative of UL9 protein binding only. Consistent with the assumption that ICP8 increases the processivity of the UL9 protein, there was no effect of ICP8 on the rate of ATP hydrolysis with (dT)15. In contrast, ATP hydrolysis with longer DNA cofactors, which allow both binding and translocation of the UL9 protein, was greatly stimulated by ICP8. Furthermore, ICP8 had an even greater stimulatory effect on ATP hydrolysis with DNA cofactors that contain secondary structure. In conclusion, the ability of ICP8 to prevent competition with challenger DNA during DNA unwinding, and to stimulate ATP hydrolysis with DNA cofactors that allow translocation, implies that it prevents dissociation of the UL9 protein from the DNA substrate and increases its processivity. In addition, ICP8 appears to facilitate translocation of the UL9 protein through regions of duplex DNA. This is probably a manifestation of the helix-destabilizing activity of ICP8 (24Boehmer P.E. Lehman I.R. J. Virol. 1993; 67: 711-715Crossref PubMed Google Scholar). Fig. 9 shows a model of how ICP8 stimulates the translocation of the UL9 protein along ssDNA and through regions of duplex DNA. Both molecules within the UL9 protein dimer (6Bruckner R.C. Crute J.J. Dodson M.S. Lehman I.R. J. Biol. Chem. 1991; 266: 2669-2674Abstract Full Text PDF PubMed Google Scholar,7Fierer D.S. Challberg M.D. J. Virol. 1992; 66: 3986-3995Crossref PubMed Google Scholar, 19Makhov A.M. Boehmer P.E. Lehman I.R. Griffith J.D. J. Mol. Biol. 1996; 258: 789-799Crossref PubMed Scopus (39) Google Scholar, 20Makhov A.M. Boehmer P.E. Lehman I.R. Griffith J.D. EMBO J. 1996; 15: 1742-1750Crossref PubMed Scopus (46) Google Scholar) make contact with ssDNA via their N-terminal ssDNA-binding regions (25Abbotts A.P. Stow N.D. J. Gen. Virol. 1995; 76: 3125-3130Crossref PubMed Scopus (18) Google Scholar), enabling it to translocate 3′ to 5′ (7Fierer D.S. Challberg M.D. J. Virol. 1992; 66: 3986-3995Crossref PubMed Google Scholar, 9Boehmer P.E. Dodson M.S. Lehman I.R. J. Biol. Chem. 1993; 268: 1220-1225Abstract Full Text PDF PubMed Google Scholar). One molecule of ICP8 is bound to the C terminus of each UL9 protein molecule (11Boehmer P.E. Lehman I.R. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 8444-8448Crossref PubMed Scopus (90) Google Scholar,12Boehmer P.E. Craigie M.C. Stow N.D. Lehman I.R. J. Biol. Chem. 1994; 269: 29329-29334Abstract Full Text PDF PubMed Google Scholar), allowing one ICP8 molecule to bind the template strand, tethering the UL9 protein to the DNA substrate, while the second molecule of ICP8 assimilates and stabilizes the unwound primer strand, thereby facilitating the translocation of the UL9 protein through regions of duplex DNA. Previous studies have shown that a site-specific cisplatin lesion impairs the DNA helicase activity of the UL9 protein (22Villani G. Pillaire M.J. Boehmer P.E. J. Biol. Chem. 1994; 269: 21676-21681Abstract Full Text PDF PubMed Google Scholar). Furthermore, it was shown that ICP8 relieved the inhibitory effect imposed by the lesion. Based on the findings in this study, it is likely that ICP8 enables the UL9 protein to bypass the lesion by tethering it to the DNA substrate, thereby preventing its dissociation. The effects of cognate and noncognate SSBs have been documented for numerous DNA helicases (reviewed in Refs. 26Kornberg A. Baker T. DNA Replication. W. H. Freeman & Co., New York1992Google Scholar, 27Hübscher U. Maga G. Podust V.N. DePamphilis M.J. DNA Replication in Eukaryotic Cells. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY1996: 525-543Google Scholar, 28Borowiec J.A. DePamphilis M.J. DNA Replication in Eukaryotic cells. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY1996: 545-574Google Scholar). E-SSB has been shown to affect the activities of E. coli PriA (29Lee M.S. Marians K.J. Proc. Natl. Acad. Sci. U. S. A. 1987; 84: 8345-8349Crossref PubMed Scopus (83) Google Scholar, 30Lasken R.S. Kornberg A. J. Biol. Chem. 1988; 263: 5512-5518Abstract Full Text PDF PubMed Google Scholar), DNA helicase IV (31Yancey-Wrona J.E. Wood E.R. George J.W. Smith K.R. Matson S.W. Eur. J. Biochem. 1992; 207: 479-485Crossref PubMed Scopus (15) Google Scholar), and Rep (32Scott J.F. Eisenberg S. Bertsch L. Kornberg A. Proc. Natl. Acad. Sci. U. S. A. 1977; 74: 193-197Crossref PubMed Scopus (134) Google Scholar, 33Yarranton G.T. Gefter M.L. Proc. Natl. Acad. Sci. U. S. A. 1979; 76: 1658-1662Crossref PubMed Scopus (149) Google Scholar) DNA helicases. Likewise, replication protein-A (RP-A) has been shown to affect several eukaryotic DNA helicases including: Saccharomyces cerevisiae Hel B (HCSB) (34Biswas E.E. Chen P.H. Biswas S.B. Biochem. 1993; 32: 13393-13398Crossref PubMed Scopus (17) Google Scholar, 35Biswas E.E. Chen P.H. Leszyk J. Biswas S.B. Biochem. Biophys. Res. Commun. 1995; 206: 850-856Crossref PubMed Scopus (19) Google Scholar), calf thymus DNA helicases A–D and F (36Thömmes P. Ferrari E. Jessberger R. Hübscher U. J. Biol. Chem. 1992; 267: 6063-6073Abstract Full Text PDF PubMed Google Scholar, 37Zhang S. Grosse F. FEBS Lett. 1992; 312: 143-146Crossref PubMed Scopus (9) Google Scholar, 38Georgaki A. Tuteja N. Sturzenegger B. Hübscher U. Nucleic Acids Res. 1994; 22: 1128-1134Crossref PubMed Scopus (17) Google Scholar), human DNA helicases isolated from HeLa cells (39Seo Y.-S. Lee S.H. Hurwitz J. J. Biol. Chem. 1991; 266: 13161-13170Abstract Full Text PDF PubMed Google Scholar, 40Seo Y.-S. Hurwitz J. J. Biol. Chem. 1993; 268: 10282-10295Abstract Full Text PDF PubMed Google Scholar), and simian virus 40 large T antigen (41Dodson M. Dean F. Bullock P. Echols H. Hurwitz J. Science. 1987; 238: 964-967Crossref PubMed Scopus (98) Google Scholar, 42Wold M. Li J. Kelly T. Proc. Natl. Acad. Sci. U. S. A. 1987; 84: 3643-3647Crossref PubMed Scopus (159) Google Scholar, 43Dornreiter I. Erdile L.F. Gilbert I.U. von Winkler D. Kelly T.J. Fanning E. EMBO J. 1992; 11: 769-776Crossref PubMed Scopus (284) Google Scholar). In HSV-1, apart from the interaction between ICP8 and the UL9 protein discussed in this report, it has also been shown that ICP8 can specifically stimulate the DNA helicase activity of the DNA helicase-primase (17Le Gac N.T. Villani G. Hoffman J.-S. Boehmer P.E. J. Biol. Chem. 1996; 271: 21645-21651Abstract Full Text Full Text PDF PubMed Scopus (51) Google Scholar, 18Falkenberg M. Bushnell D.A. Elias P. Lehman I.R. J. Biol. Chem. 1997; 272: 22766-22770Crossref PubMed Scopus (53) Google Scholar, 44Crute J.J. Lehman I.R. J. Biol. Chem. 1991; 266: 4484-4488Abstract Full Text PDF PubMed Google Scholar). In most cases, SSBs have been shown to enhance DNA unwinding by preventing nonproductive binding of the DNA helicase to the DNA substrate. Specific protein-protein interactions between DNA helicases and cognate SSBs have also been shown to increase the length of DNA unwound. However, no previous studies have addressed the exact mechanism by which an SSB stimulates DNA unwinding. Previous studies have indicated the importance of the ICP8-UL9 protein interaction for HSV-1 origin-specific DNA replication (12Boehmer P.E. Craigie M.C. Stow N.D. Lehman I.R. J. Biol. Chem. 1994; 269: 29329-29334Abstract Full Text PDF PubMed Google Scholar, 21Lee S.S.-K. Lehman I.R. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 2838-2842Crossref PubMed Scopus (51) Google Scholar). The properties of the UL9 protein and ICP8 and those of the ICP8-UL9 protein complex, described in this and previous studies (7Fierer D.S. Challberg M.D. J. Virol. 1992; 66: 3986-3995Crossref PubMed Google Scholar, 9Boehmer P.E. Dodson M.S. Lehman I.R. J. Biol. Chem. 1993; 268: 1220-1225Abstract Full Text PDF PubMed Google Scholar, 10Ruyechan W.T. J. Virol. 1983; 46: 661-666Crossref PubMed Google Scholar, 20Makhov A.M. Boehmer P.E. Lehman I.R. Griffith J.D. EMBO J. 1996; 15: 1742-1750Crossref PubMed Scopus (46) Google Scholar,21Lee S.S.-K. Lehman I.R. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 2838-2842Crossref PubMed Scopus (51) Google Scholar, 24Boehmer P.E. Lehman I.R. J. Virol. 1993; 67: 711-715Crossref PubMed Google Scholar), illustrate how this complex is suited for its predicted role of recognizing and unwinding the HSV-1 origins of DNA replication.

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