Effect of Single-stranded DNA-binding Proteins on the Helicase and Primase Activities of the Bacteriophage T7 Gene 4 Protein

Gene 4 protein (gp4) of bacteriophage T7 provides two essential functions at the T7 replication fork, primase and helicase activities. Previous studies have shown that the single-stranded DNA-binding protein of T7, encoded by gene 2.5, interacts with gp4 and modulates its multiple functions. To further characterize the interactions between gp4 and gene 2.5 protein (gp2.5), we have examined the effect of wild-type and altered gene 2.5 proteins as well as Escherichia coli single-stranded DNA-binding (SSB) protein on the ability of gp4 to synthesize primers, hydrolyze dTTP, and unwind duplex DNA. Wild-type gp2.5 and E. coli SSB protein stimulate primer synthesis and DNA-unwinding activities of gp4 at low concentrations but do not significantly affect single-stranded DNA-dependent hydrolysis of dTTP. Neither protein inhibits the binding of gp4 to single-stranded DNA. The variant gene 2.5 proteins, gp2.5-F232L and gp2.5-Δ26C, inhibit primase, dTTPase, and helicase activities proportional to their increased affinities for DNA. Interestingly, wild-type gp2.5 stimulates the unwinding activity of gp4 except at very high concentrations, whereas E. coli SSB protein is highly inhibitory at relative low concentrations. Gene 4 protein (gp4) of bacteriophage T7 provides two essential functions at the T7 replication fork, primase and helicase activities. Previous studies have shown that the single-stranded DNA-binding protein of T7, encoded by gene 2.5, interacts with gp4 and modulates its multiple functions. To further characterize the interactions between gp4 and gene 2.5 protein (gp2.5), we have examined the effect of wild-type and altered gene 2.5 proteins as well as Escherichia coli single-stranded DNA-binding (SSB) protein on the ability of gp4 to synthesize primers, hydrolyze dTTP, and unwind duplex DNA. Wild-type gp2.5 and E. coli SSB protein stimulate primer synthesis and DNA-unwinding activities of gp4 at low concentrations but do not significantly affect single-stranded DNA-dependent hydrolysis of dTTP. Neither protein inhibits the binding of gp4 to single-stranded DNA. The variant gene 2.5 proteins, gp2.5-F232L and gp2.5-Δ26C, inhibit primase, dTTPase, and helicase activities proportional to their increased affinities for DNA. Interestingly, wild-type gp2.5 stimulates the unwinding activity of gp4 except at very high concentrations, whereas E. coli SSB protein is highly inhibitory at relative low concentrations. The single-stranded DNA (ssDNA) 1The abbreviations used are: ssDNA, single-stranded DNA; gp4 and gp2.5, gene 4 protein and gene 2.5 protein, respectively; WT, wild type; SSB protein, single-stranded DNA-binding protein; nt, nucleotide. -binding protein of bacteriophage T7, encoded by gene 2.5 of the phage, is essential for T7 DNA replication and hence for growth of the phage (1Kim Y.T. Richardson C.C. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 10173-10177Crossref PubMed Scopus (60) Google Scholar). Gene 2.5 protein (gp2.5) is one of four proteins that constitute a T7 replisome capable of mediating DNA replication in which leading and lagging strand syntheses are coordinated and the size distribution of Okazaki fragments is maintained via a replication loop formed from the lagging strand (2Lee J. Chastain P.D. Kusakabe T. Griffith J.D. Richardson C.C. Mol. Cell. 1998; 1: 1001-1010Abstract Full Text Full Text PDF PubMed Scopus (124) Google Scholar, 3Lee J. Chastain P.D. Griffith J.D. Richardson C.C. J. Mol. Biol. 2002; 316: 19-34Crossref PubMed Scopus (56) Google Scholar). In addition to gp2.5 the replisome contains T7 gene 5 DNA polymerase, its processivity factor Escherichia coli thioredoxin, and the multifunctional T7 gene 4 helicase-primase or gp4 (4Richardson C.C. Cell. 1983; 33: 315-317Abstract Full Text PDF PubMed Scopus (98) Google Scholar). Gp2.5 is unique among these proteins in that it not only binds to ssDNA but also interacts with T7 DNA and gp4 Y.T. S. Richardson C.C. J. Biol. Full Text PDF PubMed Google Scholar, Y.T. Richardson C.C. J. Biol. Full Text PDF PubMed Google Scholar). the of the of gp2.5 with ssDNA and with T7 DNA have Y.T. S. Richardson C.C. J. Biol. Full Text PDF PubMed Google Scholar, S. Griffith J.D. Richardson C.C. J. Biol. Full Text Full Text PDF PubMed Scopus Google is the effect of gp2.5 on the helicase and primase activities of T7 Gp2.5 to a of proteins that are not only essential for DNA replication but also in DNA and Y.T. S. Griffith J.D. Richardson C.C. J. Biol. Full Text PDF PubMed Google Scholar, PubMed Scopus Google Scholar). for is essential for in T7 and in it S. Richardson C.C. S. Scholar, S. Griffith J.D. Richardson C.C. J. Biol. Full Text Full Text PDF PubMed Scopus Google Scholar). a of T7 gp2.5 is and to the protein of E. coli and the gene protein of bacteriophage of a gp2.5 T. Richardson C.C. T. Proc. Natl. Acad. Sci. U. S. A. PubMed Scopus Google that it contains binding among of the SSB protein E. coli SSB protein and gene and E. coli SSB protein gene protein T7 gp2.5 for T7 growth (1Kim Y.T. Richardson C.C. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 10173-10177Crossref PubMed Scopus (60) Google Scholar, Richardson C.C. J. Biol. 1998; Full Text Full Text PDF PubMed Scopus Google Scholar). the interactions of gp2.5 with ssDNA and with T7 DNA and gp4 that for have to to is the ability of of the two proteins to ssDNA with T7 gp2.5 binds to ssDNA with a E. coli SSB protein or gene protein Y.T. S. Griffith J.D. Richardson C.C. J. Biol. Full Text PDF PubMed Google Scholar). T7 DNA a primer on ssDNA with gp2.5, E. coli SSB protein, or gene protein Y.T. S. Richardson C.C. J. Biol. Full Text PDF PubMed Google Scholar). the gp4 strand in the of gene protein, it not ssDNA with gene protein Richardson C.C. J. Biol. Full Text Full Text PDF PubMed Scopus Google Scholar). by the gene 4 helicase in the of gp2.5 Richardson C.C. J. Biol. Full Text Full Text PDF PubMed Scopus Google Scholar). T7 gp2.5, E. coli SSB protein, and gene protein have of which a of studies shown are essential for the interactions in Y.T. Richardson C.C. J. Biol. Full Text PDF PubMed Google Scholar, S. Griffith J.D. Richardson C.C. J. Biol. Full Text Full Text PDF PubMed Scopus Google Scholar, Richardson C.C. J. Biol. 1998; Full Text Full Text PDF PubMed Scopus Google Scholar, U. J. J. PubMed Scopus Google Scholar, J. U. Biol. PubMed Scopus Google Scholar, J. J. Biol. Full Text PDF PubMed Google Scholar, J. Biol. Full Text Full Text PDF PubMed Scopus Google Scholar, Proc. Natl. Acad. Sci. U. S. A. PubMed Scopus Google Scholar). gp2.5, the T7 and the protein or with T7 DNA or gp4 Y.T. Richardson C.C. J. Biol. Full Text PDF PubMed Google Scholar). the of these proteins are essential for are not for the for T7 proteins in which the of E. coli SSB protein or gene protein for the of gp2.5 the growth of T7 phage gene and with gp4 and T7 DNA the proteins of SSB protein or gene protein the T7 not the growth of T7 gene 2.5 Richardson C.C. J. Biol. 1998; Full Text Full Text PDF PubMed Scopus Google Scholar). a we a of that not T7 growth T. T. Richardson C.C. J. Biol. 2002; Full Text Full Text PDF PubMed Scopus Google Scholar). studies with these altered proteins to the of that DNA binding S. Griffith J.D. Richardson C.C. J. Biol. Full Text Full Text PDF PubMed Scopus Google Scholar, Richardson C.C. J. Biol. Full Text Full Text PDF PubMed Scopus Google and S. Griffith J.D. Richardson C.C. J. Biol. Full Text Full Text PDF PubMed Scopus Google of the altered proteins to in interactions with gp4 or T7 DNA altered protein, in the a in the T. T. Richardson C.C. J. Biol. 2002; Full Text Full Text PDF PubMed Scopus Google Scholar). binds to ssDNA gp2.5 but not as as S. Griffith J.D. Richardson C.C. J. Biol. Full Text Full Text PDF PubMed Scopus Google Scholar). stimulates T7 DNA activity on ssDNA gp2.5, whereas inhibits T7 DNA the of the in interactions with the well are the of gp2.5 on the helicase and primase activities of the T7 The helicase and primase activities of gp4 in the and of the protein, Richardson C.C. Proc. Natl. Acad. Sci. U. S. A. PubMed Scopus Google Scholar). The DNA and the helicase and primase fragments have and Richardson C.C. Proc. Natl. Acad. Sci. U. S. A. PubMed Scopus Google Scholar). of the helicase S. S. Richardson C.C. T. Cell. Full Text Full Text PDF PubMed Scopus Google Scholar, T. Cell. Full Text Full Text PDF PubMed Scopus Google and primase T. Richardson C.C. T. Mol. Cell. Full Text Full Text PDF Scopus Google fragments are The helicase and primase fragments have helicase and primase to that of the wild-type as the helicase to the primase as in the protein, on the primase The multiple activities by the of which interactions with ssDNA, multiple that are for it is to the by of the by the helicase the of the protein a In the of dTTP, the gp4 as a on ssDNA Proc. Natl. Acad. Sci. U. S. A. PubMed Scopus Google Scholar, Biol. PubMed Scopus Google with a that the helicase the of the DNA to which it is Proc. Natl. Acad. Sci. U. S. A. PubMed Scopus Google Scholar). of the is on the that the helicase and primase S. S. Richardson C.C. T. Cell. Full Text Full Text PDF PubMed Scopus Google Scholar, T. Cell. Full Text Full Text PDF PubMed Scopus Google Scholar, S. S. Richardson C.C. J. Biol. Full Text Full Text PDF PubMed Scopus Google Scholar). to ssDNA, the protein from to on the a to the hydrolysis of J. Mol. Biol. 2002; PubMed Scopus Google Scholar, S. Richardson C.C. Proc. Natl. Acad. Sci. U. S. A. PubMed Scopus Google Scholar). a duplex that is a on the strand that it it unwind the Gp2.5 to ssDNA to the gp4 the of the the helicase of E. coli or the gene helicase of phage J. Biol. Full Text PDF PubMed Google Scholar, J. J. Biol. the T7 helicase not have a helicase ssDNA, the of ssDNA or of the The primase the synthesis of on ssDNA, and in are to the T7 DNA for as to DNA synthesis Richardson C.C. PubMed Scopus Google Scholar). the primase the primase the synthesis of the from and Richardson C.C. J. Biol. Full Text PDF PubMed Google Scholar). that the are or and the primers, and are S. Richardson C.C. Proc. Natl. Acad. Sci. U. S. A. PubMed Scopus Google Scholar). in is by a at the of the primase Y.T. S. Richardson C.C. J. Biol. Full Text PDF PubMed Google Scholar, Richardson C.C. J. Biol. Full Text PDF PubMed Google Scholar, Richardson C.C. Proc. Natl. Acad. Sci. U. S. A. 1993; Scopus Google Scholar). the effect of DNA on the of the primase and the synthesis of the primer are of The primase the helicase the synthesis of as well as the gp4 in the of Richardson C.C. Proc. Natl. Acad. Sci. U. S. A. 1998; PubMed Scopus Google Scholar). the primase a very low for ssDNA and for its Richardson C.C. J. Biol. Full Text Full Text PDF PubMed Scopus Google Scholar). the gp4 primase activity in the of of the ability of the helicase to to ssDNA Richardson C.C. Proc. Natl. Acad. Sci. U. S. A. 1998; PubMed Scopus Google Scholar). DNA with a of primase the primase is on by the helicase for to these Richardson C.C. Proc. Natl. Acad. Sci. U. S. A. 1998; PubMed Scopus Google Scholar). a gp2.5 have effect on primase activity via its with the helicase In a S. Griffith J.D. Richardson C.C. J. Biol. Full Text Full Text PDF PubMed Scopus Google we examined the effect of wild-type gp2.5 and of gp2.5 for their ability to to ssDNA and to with T7 DNA Wild-type gp2.5 to stimulate DNA synthesis by T7 DNA on ssDNA The ability of T7 DNA to synthesis DNA is on the of the in that the inhibits synthesis by S. Griffith J.D. Richardson C.C. J. Biol. Full Text Full Text PDF PubMed Scopus Google Scholar). In we examined the effect of gp2.5 on the multiple activities by the helicase and primase of In these we have used two altered and as well as the wild-type a in the that its binding to ssDNA not affect its with the T7 replication proteins S. Griffith J.D. Richardson C.C. J. Biol. Full Text Full Text PDF PubMed Scopus Google Scholar). In we have examined the effect of E. coli SSB protein on these to the for gp2.5 in the T7 and coli used as the to T7 gene 2.5 and to wild-type and Wild-type and gene 2.5 are from the the T7 T7 from E. coli from DNA and used in the to primase activity and and to helicase and from DNA to and used for helicase ssDNA from The used for of gene 2.5 S. Griffith J.D. Richardson C.C. J. Biol. Full Text Full Text PDF PubMed Scopus Google and their and to the DNA for helicase and to the DNA for helicase and to the DNA for helicase activity in a and DNA and from E. coli SSB protein from T7 and the primase of T7 of the and from and gp2.5 and gp2.5-F232L and as S. Griffith J.D. Richardson C.C. J. Biol. Full Text Full Text PDF PubMed Scopus Google Scholar). by T7 DNA synthesis of by gp4 by the of ssDNA or DNA a primase Richardson C.C. J. Biol. Full Text PDF PubMed Google Scholar, Richardson C.C. J. Biol. Full Text Full Text PDF PubMed Scopus Google Scholar). The 4 ssDNA or of and dTTP, and The with the of proteins for 5 at and the by the addition of gp4 or gp4 primase further at for the by the addition of of and and a the a the hydrolysis of dTTP, a to its on ssDNA Richardson C.C. J. Biol. 1983; Full Text PDF PubMed Google Scholar). The ssDNA, and The with the of protein for 5 at and the by the addition of further at for the by to a of The a The with a and The a DNA for helicase activity the of a to a ssDNA S. Richardson C.C. J. Biol. 1983; Full Text PDF PubMed Google Scholar). helicase by a to a in The helicase DNA dTTP, and The with the of protein for 5 at and the by the addition of further for at the by to a of The a the from the duplex a In a in which of ssDNA and we that is of the DNA it is that of ssDNA the helicase leading to of DNA of Gp2.5 and E. coli SSB on a gp4 for the that gp4 synthesis on ssDNA Richardson C.C. J. Biol. Full Text PDF PubMed Google Scholar). studies and also that its synthesis Richardson C.C. J. Biol. 1998; Full Text Full Text PDF PubMed Scopus Google Scholar). In that E. coli SSB protein synthesis whereas protein synthesis by Richardson C.C. J. Biol. 1998; Full Text Full Text PDF PubMed Scopus Google Scholar). To further characterize these we examined the ability of gp2.5 and E. coli SSB protein to affect primer synthesis at concentrations. activity ssDNA with gp2.5 or E. coli SSB with the gp2.5 and E. coli SSB protein synthesis studies have shown that gp2.5 is to ssDNA Richardson C.C. J. Biol. Full Text Full Text PDF PubMed Scopus Google Scholar). In 4 ssDNA, we used a gp2.5 of which is to ssDNA on the that one gp2.5 on ssDNA Y.T. S. Griffith J.D. Richardson C.C. J. Biol. Full Text PDF PubMed Google Scholar). E. coli SSB protein a for ssDNA relative to gp2.5 Y.T. S. Griffith J.D. Richardson C.C. J. Biol. Full Text PDF PubMed Google Scholar). Interestingly, with the of gp2.5 to of the E. coli SSB protein synthesis at of To the effect of in the DNA we used a only one primase as a to primer the of protein to the ssDNA in the with the with ssDNA, gp2.5 and E. coli SSB protein the primase activity of gp4 at low concentrations of Gp2.5 and E. coli SSB on of to of primer synthesis is the of gp4 the ssDNA To proteins affect the ability of gp4 to ssDNA we used a of dTTP, T. J. Richardson C.C. J. 1998; PubMed Scopus Google Scholar). Previous studies have shown that the binding of gp4 to ssDNA and but not for on ssDNA T. J. Richardson C.C. J. 1998; PubMed Scopus Google Scholar). gp4 a or in a in the of gp2.5 or E. coli SSB protein in of the a synthesis The of on the and The or of gp2.5 or E. coli SSB protein effect on synthesis on the in the of primer synthesis and for the and that the of gp2.5 E. coli SSB protein inhibits the binding of gp4 to the ssDNA and the synthesis of To the ability of gp4 to and primer synthesis on a the with gp2.5 or E. coli SSB shown in gp4 in the of well the with gp2.5 or E. coli SSB The of gp4 in the of is To of gp4 is for we the for In the of the is a in primer synthesis The primase of gp4 the helicase is for primer synthesis The from its to to ssDNA via Richardson C.C. Proc. Natl. Acad. Sci. U. S. A. 1998; PubMed Scopus Google Scholar). that gp4 and the primase have the in the of gp2.5 or E. coli SSB of Gp2.5 and E. coli SSB on of gp4 on ssDNA is by the hydrolysis of T. J. Richardson C.C. J. 1998; PubMed Scopus Google Scholar). The on ssDNA is essential for helicase and it also the primase to primase S. Richardson C.C. Proc. Natl. Acad. Sci. U. S. A. PubMed Scopus Google Scholar). To the effect of gp2.5 and E. coli SSB protein on activity of we activity at concentrations of the two proteins with ssDNA and In the of gp2.5 we used is to ssDNA Y.T. S. Griffith J.D. Richardson C.C. J. Biol. Full Text PDF PubMed Google Scholar). Gp2.5 stimulates the activity at concentrations and E. coli SSB protein, on the to a at or and Interestingly, the of gp2.5 and E. coli SSB protein to the ssDNA is and the of the protein on activity the ssDNA is with the proteins of Gp2.5 and E. coli SSB on the unwinding ability of gp4 is by the of proteins, helicase activity in the and of gp2.5 and E. coli SSB The DNA used in the unwinding is shown in The DNA of a duplex DNA with a and a and nt, The provides a for gp4 to as a and the gp4 from the duplex DNA J. Biol. Full Text Full Text PDF PubMed Scopus Google Scholar). the with one of the proteins, gp4 to the unwinding Gp2.5 stimulates the unwinding activity at 5 and The effect is between and which it a inhibitory In E. coli SSB protein only a effect on unwinding activity at a very low is the inhibitory effect at the high of SSB protein, in a at and Neither protein unwinding activity not of of Gp2.5 on of Previous studies have shown that the of gp2.5 is for its with and the T7 replication T7 or the or do not or with T7 DNA or T7 gp4 Y.T. S. Griffith J.D. Richardson C.C. J. Biol. Full Text PDF PubMed Google Scholar, T. T. Richardson C.C. J. Biol. 2002; Full Text Full Text PDF PubMed Scopus Google Scholar). variant have a for ssDNA relative to the gp2.5 S. Griffith J.D. Richardson C.C. J. Biol. Full Text Full Text PDF PubMed Scopus Google Scholar, Y.T. S. Griffith J.D. Richardson C.C. J. Biol. Full Text PDF PubMed Google Scholar, Richardson C.C. J. Biol. Full Text Full Text PDF PubMed Scopus Google Scholar). also inhibit DNA synthesis by T7 DNA on ssDNA, and shown to inhibit the primase activity of gp4 Richardson C.C. J. Biol. 1998; Full Text Full Text PDF PubMed Scopus Google Scholar). altered gp2.5, a in the S. Griffith J.D. Richardson C.C. J. Biol. Full Text Full Text PDF PubMed Scopus Google Scholar, T. T. Richardson C.C. J. Biol. 2002; Full Text Full Text PDF PubMed Scopus Google Scholar). to a gp2.5 that binds to ssDNA relative to gp2.5 S. Griffith J.D. Richardson C.C. J. Biol. Full Text Full Text PDF PubMed Scopus Google Scholar). in to the gp2.5-F232L and its interactions with T7 DNA S. Griffith J.D. Richardson C.C. J. Biol. Full Text Full Text PDF PubMed Scopus Google Scholar). In gp2.5-F232L stimulates T7 DNA activity on ssDNA S. Griffith J.D. Richardson C.C. J. Biol. Full Text Full Text PDF PubMed Scopus Google Scholar). have used these altered to characterize further the interactions of gp2.5 with In the shown in we have the of and gp2.5-F232L on the activity of Wild-type gp2.5 is for In to the with wild-type gp2.5, of the variant activity is the is with the of the protein, and the activity is at concentrations The of gp2.5 to the ssDNA is inhibits activity at concentrations and the activity is by at the of is a of activity at the low concentrations, the with wild-type of of Gp2.5 on DNA by effect of and gp2.5-F232L on the unwinding activity of gp4 examined the DNA and unwinding in wild-type gp2.5 a of unwinding activity at concentrations to in of its of activity and hence unwinding by gp4 and on the in a to wild-type gp2.5 with the that the of and of of Gp2.5 on by in the with gp2.5 and E. coli SSB protein, we examined the effect of and gp2.5-F232L on synthesis by gp4 on a a primase in wild-type gp2.5 a effect on primase and at concentrations a with Richardson C.C. J. Biol. 1998; Full Text Full Text PDF PubMed Scopus Google is inhibitory to primase whereas gp2.5-F232L wild-type gp2.5 with a at the concentrations synthesis by gp4 the of a primase and the of S. Richardson C.C. Proc. Natl. Acad. Sci. U. S. A. PubMed Scopus Google Scholar, Richardson C.C. J. Biol. Full Text PDF PubMed Google Scholar). The of the is by the of the primase Y.T. S. Richardson C.C. J. Biol. Full Text PDF PubMed Google Scholar, Richardson C.C. J. Biol. Full Text PDF PubMed Google Scholar, Richardson C.C. Proc. Natl. Acad. Sci. U. S. A. 1993; Scopus Google Scholar). of these on ssDNA the helicase activity of which not only the primase to the but also the primase to the DNA via the binding of the helicase to the ssDNA Richardson C.C. Proc. Natl. Acad. Sci. U. S. A. 1998; PubMed Scopus Google Scholar). the variant inhibit the of gp4 on ssDNA as shown at a of of primase activity in is the of the of the helicase to the primase at its To these we have used the primase of gp4 the helicase shown in gp2.5-F232L affect on synthesis by the primase is to that synthesis by the primase is that it is to the helicase a of primase relative to that used for the protein in A. the activity of the primase is by the of these variant The gene 4 helicase-primase of bacteriophage T7 multiple functions at the T7 replication The primase the synthesis of on the lagging strand in are to T7 DNA for as T. Richardson C.C. J. Biol. Full Text Full Text PDF PubMed Scopus Google Scholar, J. S. Richardson C.C. T. J. Biol. Full Text Full Text PDF PubMed Scopus Google Scholar, S. Richardson C.C. Proc. Natl. Acad. Sci. U. S. A. PubMed Scopus Google Scholar). The helicase is for the to of the protein on ssDNA as well as the unwinding of duplex DNA to single-stranded for the leading strand DNA T. Cell. Full Text Full Text PDF PubMed Scopus Google Scholar, S. Richardson C.C. J. Biol. 1983; Full Text PDF PubMed Google Scholar, PubMed Scopus Google Scholar, J. Biol. Full Text PDF PubMed Google Scholar). Previous studies have shown that gp2.5 is essential for the of leading and lagging strand DNA synthesis at a replication (2Lee J. Chastain P.D. Kusakabe T. Griffith J.D. Richardson C.C. Mol. Cell. 1998; 1: 1001-1010Abstract Full Text Full Text PDF PubMed Scopus (124) Google Scholar, 3Lee J. Chastain P.D. Griffith J.D. Richardson C.C. J. Mol. Biol. 2002; 316: 19-34Crossref PubMed Scopus (56) Google Scholar). gp2.5 is to with two of the T7 T7 DNA Y.T. S. Richardson C.C. J. Biol. Full Text PDF PubMed Google and gp4 Y.T. Richardson C.C. J. Biol. Full Text PDF PubMed Google as well as to their ssDNA it is to the of these interactions on their In a S. Griffith J.D. Richardson C.C. J. Biol. Full Text Full Text PDF PubMed Scopus Google we that interactions of gp2.5 with T7 DNA are for its ability to DNA synthesis on In the we have examined the effect of gp2.5 on the primase and helicase activities of studies have by a of wild-type gp2.5 with altered gp2.5 proteins and with E. coli SSB E. coli SSB protein for T7 gp2.5 phage T7 and it a for ssDNA relative to gp2.5 (1Kim Y.T. Richardson C.C. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 10173-10177Crossref PubMed Scopus (60) Google Scholar). The two altered gp2.5 proteins, and have in their a that is essential for the interactions and to E. coli SSB protein, a for ssDNA with wild-type gp2.5 S. Griffith J.D. Richardson C.C. J. Biol. Full Text Full Text PDF PubMed Scopus Google Scholar). The ability of gp4 to ssDNA and to the DNA strand by the hydrolysis of S. Richardson C.C. J. Biol. 1983; Full Text PDF PubMed Google Scholar). we that the binding of gp2.5 or E. coli SSB protein to ssDNA effect on the ability of gp4 to on In the is very inhibitory for also a at concentrations. E. coli SSB protein the for ssDNA not inhibit it that the inhibitory of from its to with studies with proteins that among the T7, and E. coli proteins Richardson C.C. J. Biol. 1998; Full Text Full Text PDF PubMed Scopus Google Scholar). In it that gp4 ssDNA at the replication it is with T7 DNA as it duplex DNA with the ssDNA The effect of the proteins on unwinding of duplex DNA their on are E. coli SSB protein and are very inhibitory at low concentrations. wild-type gp2.5 and gp2.5-F232L are inhibitory at high concentrations, a for which we have it from the ability of gp2.5 to leading to of the DNA their by helicase of the effect of gp2.5 on primase activity is to activity is by the binding of gp4 via its helicase to ssDNA Richardson C.C. Proc. Natl. Acad. Sci. U. S. A. 1998; PubMed Scopus Google Scholar). In the ability of gp4 to on ssDNA, via its helicase it to primase on a DNA effect of gp2.5 on the activity of gp2.5 in primase activity as by the of a primase only the primase of gp4 or the of very that is not a to primer the DNA not the binding of the gp4 to ssDNA via a that stimulates primase activity significantly Richardson C.C. Proc. Natl. Acad. Sci. U. S. A. 1998; PubMed Scopus Google Scholar). that gp4 gp2.5 or E. coli SSB ssDNA and ssDNA, studies have shown that primase to the binding for helicase of these that gp2.5 or E. coli SSB protein stimulates primer synthesis at low concentrations. and gp2.5-F232L are inhibitory to DNA primase a that by their of of gp4 is by the of the DNA-dependent activity by these binding proteins we have shown that a for ssDNA protein and gp2.5-F232L for ssDNA that is between of gp2.5 and S. Griffith J.D. Richardson C.C. J. Biol. Full Text Full Text PDF PubMed Scopus Google Scholar). In we that of primase activity by at a of whereas gp2.5-F232L inhibits primer synthesis at a of gp2.5 not inhibit primer synthesis the the variant inhibit gp4 primase activity proportional to their increased affinities for DNA. altered inhibit and helicase activities of gp4 and the with concentrations of 5 and to with the DNA binding activity of the gp2.5 protein as shown S. Griffith J.D. Richardson C.C. J. Biol. Full Text Full Text PDF PubMed Scopus Google Scholar, T. T. Richardson C.C. J. Biol. 2002; Full Text Full Text PDF PubMed Scopus Google that gp2.5-F232L not the growth of phage gene the it that the in the of altered protein for ssDNA and its with T7 DNA its to T7 S. Griffith J.D. Richardson C.C. J. Biol. Full Text Full Text PDF PubMed Scopus Google that gp2.5-F232L strand synthesis by T7 DNA the that a Okazaki fragments on the lagging strand or with the multiple that phage The that gp2.5-F232L inhibits the primase activity of gp4 provides for its on that gp4 strand in the of gp2.5 but not it that T7 gp4 is to ssDNA with Richardson C.C. J. Biol. Full Text Full Text PDF PubMed Scopus Google Scholar). that gp4 ssDNA with E. coli SSB protein and with gp2.5 proteins that have altered DNA binding that not is to that E. coli SSB protein is in the T7 DNA replication and the T7 replication have to or to of the of E. coli SSB the studies on strand activity not among or unwinding of duplex DNA. The of of E. coli SSB protein in T7 as SSB protein not for gp2.5 in T7 DNA and it not with T7 DNA E. coli SSB protein stimulates T7 DNA activity on ssDNA gp2.5 S. Griffith J.D. Richardson C.C. J. Biol. Full Text Full Text PDF PubMed Scopus Google Scholar, Y.T. S. Griffith J.D. Richardson C.C. J. Biol. Full Text PDF PubMed Google Scholar, Richardson C.C. J. Biol. 1998; Full Text Full Text PDF PubMed Scopus Google and it gene 4 primase activity to T7 gp2.5 in the not by E. coli SSB gp2.5 and activity is essential for and in Y.T. S. Richardson C.C. J. Biol. Full Text PDF PubMed Google Scholar). the E. coli SSB protein that to T7 DNA E. coli SSB protein T7 DNA to synthesis J. Biol. Full Text PDF PubMed Google Scholar, J. Biol. Full Text Full Text PDF PubMed Scopus Google and as shown in the it inhibits unwinding of duplex DNA by The significantly binding of E. coli SSB protein to ssDNA that SSB protein to ssDNA relative to is that interactions between gp2.5 and the T7 DNA replication proteins the replication to or E. coli SSB of DNA. and for the and for very and for and for on helicase