The DNA Binding Domain of the Gene 2.5 Single-stranded DNA-binding Protein of Bacteriophage T7
2003; Elsevier BV; Volume: 278; Issue: 9 Linguagem: Inglês
10.1074/jbc.m210605200
ISSN1083-351X
AutoresEdel M. Hyland, Lisa F. Rezende, Charles C. Richardson,
Tópico(s)DNA Repair Mechanisms
ResumoGene 2.5 of bacteriophage T7 encodes a single-stranded DNA-binding protein that is essential for viral survival. Its crystal structure reveals a conserved oligosaccharide/oligonucleotide binding fold predicted to interact with single-stranded DNA. However, there is no experimental evidence to support this hypothesis. Recently, we reported a genetic screen for lethal mutations in gene 2.5 that we are using to identify functional domains of the gene 2.5 protein. This screen uncovered a number of mutations that led to amino acid substitutions in the proposed DNA binding domain. Three variant proteins, gp2.5-Y158C, gp2.5-K152E, and gp2.5-Y111C/Y158C, exhibit a decrease in binding affinity for oligonucleotides. A fourth, gp2.5-K109I, exhibits an altered mode of binding single-stranded DNA. A carboxyl-terminal truncation of gene 2.5 protein, gp2.5-Δ26C, binds single-stranded DNA 10-fold more tightly than the wild-type protein. The three altered proteins defective in single-stranded DNA binding cannot mediate the annealing of homologous DNA, whereas gp2.5-Δ26C mediates the reaction more effectively than does wild-type. Gp2.5-K109I retains this annealing ability, albeit slightly less efficiently. With the exception of gp2.5-Δ26C, all variant proteins form dimers in solution and physically interact with T7 DNA polymerase. Gene 2.5 of bacteriophage T7 encodes a single-stranded DNA-binding protein that is essential for viral survival. Its crystal structure reveals a conserved oligosaccharide/oligonucleotide binding fold predicted to interact with single-stranded DNA. However, there is no experimental evidence to support this hypothesis. Recently, we reported a genetic screen for lethal mutations in gene 2.5 that we are using to identify functional domains of the gene 2.5 protein. This screen uncovered a number of mutations that led to amino acid substitutions in the proposed DNA binding domain. Three variant proteins, gp2.5-Y158C, gp2.5-K152E, and gp2.5-Y111C/Y158C, exhibit a decrease in binding affinity for oligonucleotides. A fourth, gp2.5-K109I, exhibits an altered mode of binding single-stranded DNA. A carboxyl-terminal truncation of gene 2.5 protein, gp2.5-Δ26C, binds single-stranded DNA 10-fold more tightly than the wild-type protein. The three altered proteins defective in single-stranded DNA binding cannot mediate the annealing of homologous DNA, whereas gp2.5-Δ26C mediates the reaction more effectively than does wild-type. Gp2.5-K109I retains this annealing ability, albeit slightly less efficiently. With the exception of gp2.5-Δ26C, all variant proteins form dimers in solution and physically interact with T7 DNA polymerase. single-stranded DNA oligosaccharide/oligonucleotide binding fold nucleotide dithiothreitol gene product Single-stranded DNA (ssDNA)1-binding proteins lack sequence specificity and bind ssDNA with a higher affinity than they bind double-stranded DNA or RNA (1Chase J.W. Williams K.R. Annu. Rev. Biochem. 1986; 55: 103-136Crossref PubMed Scopus (444) Google Scholar). Primarily, ssDNA-binding proteins function to bind any exposed regions of ssDNA in cells, forming a protective coat around the reactive bases and thus restricting the formation of secondary structures. However, their role is not restricted to extending and protecting DNA in that they also physically and functionally interact with other replication proteins. Bacteriophage T7 encodes its own ssDNA-binding protein, the product of gene 2.5. Gene 2.5 protein is essential for phage survival (2Kim Y.T. Richardson C.C. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 10173-10177Crossref PubMed Scopus (58) Google Scholar) and plays multiple roles in DNA replication, recombination, and repair (2Kim Y.T. Richardson C.C. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 10173-10177Crossref PubMed Scopus (58) Google Scholar, 3Reuben R.C. Gefter M.L. Proc. Natl. Acad. Sci. U. S. A. 1973; 70: 1846-1850Crossref PubMed Scopus (49) Google Scholar, 4Scherzinger E. Litfin F. Jost E. Mol. Gen. Genet. 1973; 123: 247-262Crossref PubMed Scopus (40) Google Scholar, 5Araki H. Ogawa H. Virology. 1981; 111: 509-515Crossref PubMed Scopus (18) Google Scholar, 6Araki H. Ogawa H. Mol. Gen. Genet. 1981; 183: 66-73Crossref PubMed Scopus (15) Google Scholar, 7Nakai H. Richardson C.C. J. Biol. Chem. 1988; 263: 9831-9839Abstract Full Text PDF PubMed Google Scholar, 8Kim 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, 9Kim Y.T. Tabor S. Churchich J.E. Richardson C.C. J. Biol. Chem. 1992; 267: 15032-15040Abstract Full Text PDF PubMed Google Scholar, 10Kong D. Nossal N.G. Richardson C.C. J. Biol. Chem. 1997; 272: 8380-8387Abstract Full Text Full Text PDF PubMed Scopus (44) Google Scholar, 11Kong D. Richardson C.C. EMBO J. 1996; 15: 2010-2019Crossref PubMed Scopus (51) Google Scholar, 12Yu M. Masker W. J. Bacteriol. 2001; 183: 1862-1869Crossref PubMed Scopus (12) Google Scholar). Gene 2.5 protein interacts directly with both the T7 DNA polymerase (9Kim Y.T. Tabor S. Churchich J.E. Richardson C.C. J. Biol. Chem. 1992; 267: 15032-15040Abstract Full Text PDF PubMed Google Scholar) and the gene 4 helicase/primase (7Nakai H. Richardson C.C. J. Biol. Chem. 1988; 263: 9831-9839Abstract Full Text PDF PubMed Google Scholar), stimulating the activity of each protein. Presumably these interactions explain why coordination of leading and lagging strand synthesis in vitro is dependent upon gene 2.5 protein (13Lee J. Chastain 2nd, 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). Furthermore, gene 2.5 protein facilitates homologous DNA base pairing, 2S. Tabor and C. C. Richardson, unpublished data. 2S. Tabor and C. C. Richardson, unpublished data. a process that is important during viral recombination (10Kong D. Nossal N.G. Richardson C.C. J. Biol. Chem. 1997; 272: 8380-8387Abstract Full Text Full Text PDF PubMed Scopus (44) Google Scholar, 11Kong D. Richardson C.C. EMBO J. 1996; 15: 2010-2019Crossref PubMed Scopus (51) Google Scholar, 14Kong D. Griffith J.D. Richardson C.C. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 2987-2992Crossref PubMed Scopus (17) Google Scholar) and in the repair of double-stranded breaks in the T7 chromosome (12Yu M. Masker W. J. Bacteriol. 2001; 183: 1862-1869Crossref PubMed Scopus (12) Google Scholar).Despite functional similarity with other ssDNA-binding proteins, namely the Escherichia coli SSB protein and the bacteriophage T4 gene 32 protein, T7 gene 2.5 protein has no sequence homology with these proteins (15Prasad B.V. Chiu W. J. Mol. Biol. 1987; 193: 579-584Crossref PubMed Scopus (55) Google Scholar, 16Hollis T. Stattel J.M. Walther D.S. Richardson C.C. Ellenberger T. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 9557-9562Crossref PubMed Scopus (78) Google Scholar). Furthermore, these proteins cannot substitute for gene 2.5 protein in vivo (2Kim Y.T. Richardson C.C. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 10173-10177Crossref PubMed Scopus (58) Google Scholar, 17Kong D. Richardson C.C. J. Biol. Chem. 1998; 273: 6556-6564Abstract Full Text Full Text PDF PubMed Scopus (29) Google Scholar). The mode of binding of gene 2.5 protein to ssDNA differs from that of E. coli SSB and T4 gene 32 protein. Using a fluorescence based study gene 2.5 protein was found to have a binding constant for ssDNA binding of 1.2 × 106m−1 (8Kim 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) a value that is less than one-tenth the affinity exhibited by E. coli SSB (18Lohman T.M. Ferrari M.E. Annu. Rev. Biochem. 1994; 63: 527-570Crossref PubMed Scopus (523) Google Scholar) and T4 gene 32 protein (19Lohman T.M. Biochemistry. 1984; 23: 4656-4665Crossref PubMed Scopus (30) Google Scholar, 20Lohman T.M. Biochemistry. 1984; 23: 4665-4675Crossref PubMed Scopus (29) Google Scholar). In addition, gene 2.5 protein binds ssDNA with limited, if any, cooperativity (8Kim 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). Kimet al. (8Kim 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) reported that gene 2.5 protein bound to ssDNA with a stoichiometry of 7 nucleotides per monomer of protein, although it is not known if gene 2.5 protein binds to ssDNA as a monomer or dimer.In the absence of DNA, gene 2.5 protein aggregates to form a stable homodimer in solution (8Kim 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). Dimer formation is postulated to be dependent upon the interactions of its highly acidic carboxyl terminus (21Kim Y.T. Richardson C.C. J. Biol. Chem. 1994; 269: 5270-5278Abstract Full Text PDF PubMed Google Scholar). Its association with the other replication proteins is also facilitated by its carboxyl-terminal amino acids (13Lee J. Chastain 2nd, 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, 21Kim Y.T. Richardson C.C. J. Biol. Chem. 1994; 269: 5270-5278Abstract Full Text PDF PubMed Google Scholar). A similar role has also been shown for the acidic carboxyl-terminal tail found in the E. coli SSB protein (22Williams K.R. Spicer E.K. LoPresti M.B. Guggenheimer R.A. Chase J.W. J. Biol. Chem. 1983; 258: 3346-3355Abstract Full Text PDF PubMed Google Scholar) and the bacteriophage T4 gene 32 protein (23Burke R.L. Alberts B.M. Hosoda J. J. Biol. Chem. 1980; 255: 11484-11493Abstract Full Text PDF PubMed Google Scholar, 24Ishmael F.T. Alley S.C. Benkovic S.J. J. Biol. Chem. 2001; 276: 25236-25242Abstract Full Text Full Text PDF PubMed Scopus (47) Google Scholar).The recently solved crystal structure of a carboxyl-terminal truncation of gene 2.5 protein to a resolution of 1.9 Å (16Hollis T. Stattel J.M. Walther D.S. Richardson C.C. Ellenberger T. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 9557-9562Crossref PubMed Scopus (78) Google Scholar) revealed a core that consists of a conserved oligosaccharide/oligonucleotide binding fold (OB fold) (Fig. 1), a structure common to other ssDNA-binding proteins (25Murzin A.G. EMBO J. 1993; 12: 861-867Crossref PubMed Scopus (762) Google Scholar). As the name suggests this fold is found in proteins that bind either ssDNA such as E. coli SSB protein (26Raghunathan S. Ricard C.S. Lohman T.M. Waksman G. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 6652-6657Crossref PubMed Scopus (191) Google Scholar, 27Raghunathan S. Kozlov A.G. Lohman T.M. Waksman G. Nat. Struct. Biol. 2000; 7: 648-652Crossref PubMed Scopus (358) Google Scholar), human mitochondrial SSB protein (28Webster G. Genschel J. Curth U. Urbanke C. Kang C. Hilgenfeld R. FEBS Lett. 1997; 411: 313-316Crossref PubMed Scopus (62) Google Scholar, 29Yang C. Curth U. Urbanke C. Kang C. Nat. Struct. Biol. 1997; 4: 153-157Crossref PubMed Scopus (148) Google Scholar), all three subunits of human replication protein A (30Bochkarev A. Pfuetzner R.A. Edwards A.M. Frappier L. Nature. 1997; 385: 176-181Crossref PubMed Scopus (467) Google Scholar, 31Bochkareva E. Frappier L. Edwards A.M. Bochkarev A. J. Biol. Chem. 1998; 273: 3932-3936Abstract Full Text Full Text PDF PubMed Scopus (96) Google Scholar, 32Bochkarev A. Bochkareva E. Frappier L. Edwards A.M. EMBO J. 1999; 18: 4498-4504Crossref PubMed Scopus (149) Google Scholar), and staphlocococcal nuclease (33Alexandrescu A.T. Gittis A.G. Abeygunawardana C. Shortle D. J. Mol. Biol. 1995; 250: 134-143Crossref PubMed Scopus (46) Google Scholar) or oligosaccharides as found in E. coli heat liable enterotoxin (34Sixma T.K. Pronk S.E. Kalk K.H. van Zanten B.A. Berghuis A.M. Hol W.G. Nature. 1992; 355: 561-564Crossref PubMed Scopus (188) Google Scholar). The OB fold is comprised of a five-stranded anti-parallel β barrel, capped with an α helix. The location of this helix varies among DNA-binding proteins. In gene 2.5 protein this helix is found between the second and third strands (16Hollis T. Stattel J.M. Walther D.S. Richardson C.C. Ellenberger T. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 9557-9562Crossref PubMed Scopus (78) Google Scholar), whereas in both human replication protein A and E. coli SSB protein it connects the third and forth strands (27Raghunathan S. Kozlov A.G. Lohman T.M. Waksman G. Nat. Struct. Biol. 2000; 7: 648-652Crossref PubMed Scopus (358) Google Scholar, 30Bochkarev A. Pfuetzner R.A. Edwards A.M. Frappier L. Nature. 1997; 385: 176-181Crossref PubMed Scopus (467) Google Scholar). In gene 2.5 protein loop extensions from two of the β sheets form a prominent groove on the surface of the fold. This groove most likely represents the ssDNA interface and in Fig. 1 ssDNA is modeled into the crystal structure along this position. Located within this groove of gene 2.5 protein are two aromatic residues, tyrosine 111 and tyrosine 158. These aromatic residues, in addition to the adjacent β stands and their connecting loops comprise an evolutionarily conserved trinucleotide binding motif (16Hollis T. Stattel J.M. Walther D.S. Richardson C.C. Ellenberger T. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 9557-9562Crossref PubMed Scopus (78) Google Scholar) that binds three nucleotides in an orientation analogous with other ssDNA-binding proteins (27Raghunathan S. Kozlov A.G. Lohman T.M. Waksman G. Nat. Struct. Biol. 2000; 7: 648-652Crossref PubMed Scopus (358) Google Scholar, 30Bochkarev A. Pfuetzner R.A. Edwards A.M. Frappier L. Nature. 1997; 385: 176-181Crossref PubMed Scopus (467) Google Scholar). A number of basic residues (Lys3, Arg35, Lys107, Lys109, Lys150, and Lys152) lie in proximity to this trinucleotide binding motif forming a positively charged cleft, suggesting a role in the interaction with ssDNA (16Hollis T. Stattel J.M. Walther D.S. Richardson C.C. Ellenberger T. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 9557-9562Crossref PubMed Scopus (78) Google Scholar). However, prior to the current study, there was no direct evidence that implicates these residues in binding ssDNA.In the current study we have sought experimental evidence to support our hypothesis that the DNA binding domain lies within the OB fold of gene 2.5 protein. A previously reported random mutagenesis screen of gene 2.5 (35Rezende L.F. Hollis T. Ellenberger T. Richardson C.C. J. Biol. Chem. 2002; 277: 50643-50653Abstract Full Text Full Text PDF PubMed Scopus (22) Google Scholar) uncovered a number of lethal mutations that lead to alterations in amino acids that were predicted by the crystal structure to interact with ssDNA. Here we show that both the highly conserved aromatic residue Tyr158, one component of the trinucleotide binding motif, and the positively charged Lys152, flanking this motif, are required for the interaction with ssDNA. In addition we provide evidence for the involvement of Lys109 in the protein-DNA interaction. Finally, we show that the carboxyl-terminal tail deleted protein, gp2.5-Δ26C, binds DNA with a greater affinity than the wild-type protein.DISCUSSIONThere is a significant lack of knowledge on the structure-function relationship of the product of bacteriophage T7 gene 2.5, a ssDNA-binding protein. In the current study we sought to define the DNA binding domain of gp2.5. This study provides the first experimental evidence for the identity of the DNA binding domain of gp2.5. In a separate report (35Rezende L.F. Hollis T. Ellenberger T. Richardson C.C. J. Biol. Chem. 2002; 277: 50643-50653Abstract Full Text Full Text PDF PubMed Scopus (22) Google Scholar) we describe a genetic screen for lethal mutations in bacteriophage T7 gene 2.5. By examining the crystal structure of the protein (16Hollis T. Stattel J.M. Walther D.S. Richardson C.C. Ellenberger T. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 9557-9562Crossref PubMed Scopus (78) Google Scholar) we noted that a subset of these generated mutations lay in the postulated ssDNA binding domain. In this study we have biochemically characterized these altered gene 2.5 proteins and show that two of them, gp2.5-Y158C and gp2.5-K152E, are indeed defective in their interaction with ssDNA. A third, gp2.5-K109I, appears to interact differently with ssDNA when assessed by an electrophoretic mobility shift assay. We feel confident that the defective phenotypes do not arise from a misfolding of the protein as they physically interact with both T7 DNA polymerase and the gene 4 protein, a helicase/primase. In addition, all four proteins form dimers in a manner similar to the wild-type protein.At the onset of these studies the amino acids involved in the interaction of gene 2.5 protein with ssDNA were unknown. Previous studies on other ssDNA-binding proteins have shown that aromatic residues have the potential to intercalate between the nucleic acid bases thus stabilizing the protein-ssDNA interaction (27Raghunathan S. Kozlov A.G. Lohman T.M. Waksman G. Nat. Struct. Biol. 2000; 7: 648-652Crossref PubMed Scopus (358) Google Scholar, 41Khamis M.I. Casas-Finet J.R. Maki A.H. Murphy J.B. Chase J.W. FEBS Lett. 1987; 211: 155-159Crossref PubMed Scopus (25) Google Scholar, 42Khamis M.I. Casas-Finet J.R. Maki A.H. Murphy J.B. Chase J.W. J. Biol. Chem. 1987; 262: 10938-10945Abstract Full Text PDF PubMed Google Scholar, 43Shamoo Y. Friedman A.M. Parsons M.R. Konigsberg W.H. Steitz T.A. Nature. 1995; 376: 362-366Crossref PubMed Scopus (221) Google Scholar). In the E. coli SSB protein for example mutational studies have implicated phenylalanine 60 (Phe60) and tyrptophan 54 (Trp54) in binding ssDNA (44Casas-Finet J.R. Khamis M.I. Maki A.H. Chase J.W. FEBS Lett. 1987; 220: 347-352Crossref PubMed Scopus (55) Google Scholar, 45Ferrari M.E. Fang J. Lohman T.M. Biophys. Chem. 1997; 64: 235-251Crossref PubMed Scopus (20) Google Scholar, 46Curth U. Greipel J. Urbanke C. Maass G. Biochemistry. 1993; 32: 2585-2591Crossref PubMed Scopus (47) Google Scholar). Similarly, in T4 gene 32, protein site-directed mutagenesis has identified numerous tyrosine residues necessary for the proteins interaction with ssDNA (47Shamoo Y. Ghosaini L.R. Keating K.M. Williams K.R. Sturtevant J.M. Konigsberg W.H. Biochemistry. 1989; 28: 7409-7417Crossref PubMed Scopus (33) Google Scholar, 48Pan T. King G.C. Coleman J.E. Biochemistry. 1989; 28: 8833-8839Crossref PubMed Scopus (11) Google Scholar). T7 gene 2.5 protein possesses one such structurally conserved aromatic residue found in the OB fold. Based on our observation that the lethal substitution Y158C gives rise to a gene 2.5 protein that has a higher dissociation constant for ssDNA than that of wild-type gene 2.5 protein, we can infer that this residue plays an essential role in binding to ssDNA. Similarly, the altered gp2.5-Y111C/Y158C is also defective in binding ssDNA although the binding constant is similar to that of gp2.5-Y158C alone, implying that tyrosine 111 is not essential for ssDNA binding. This result was unexpected as an aromatic residue is conserved at this site among other ssDNA-binding proteins, specifically phenylalanine 60 in E. coli SSB protein and phenylalanine 90 in human replication protein A protein (16Hollis T. Stattel J.M. Walther D.S. Richardson C.C. Ellenberger T. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 9557-9562Crossref PubMed Scopus (78) Google Scholar).Basic residues, which can make electrostatic contacts with the negatively charged phosphates in ssDNA, are also likely candidates for DNA binding. Lysine residues have been shown to be involved in the ability of E. coli SSB protein to bind ssDNA (27Raghunathan S. Kozlov A.G. Lohman T.M. Waksman G. Nat. Struct. Biol. 2000; 7: 648-652Crossref PubMed Scopus (358) Google Scholar, 49Chen J. Smith D.L. Griep M.A. Protein Sci. 1998; 7: 1781-1788Crossref PubMed Scopus (23) Google Scholar). Indeed we have shown that substituting lysine 152 with glutamic acid weakens its binding to ssDNA, as this variant protein exhibits a 10-fold decrease in its affinity for ssDNA. Interestingly, the protein·DNA complex resulting from this interaction has difficulty adopting the slower mobility complex at concentrations comparable with wild-type gene 2.5 protein, as discussed in more detail below. Perhaps Lys152 is involved in an interaction of gene 2.5 with ssDNA at higher concentrations, and this interaction leads to the higher order structure in a manner similar to the E. coli SSB protein, which demonstrates distinct binding modes at different protein concentrations (18Lohman T.M. Ferrari M.E. Annu. Rev. Biochem. 1994; 63: 527-570Crossref PubMed Scopus (523) Google Scholar). In addition, the side chain of Lys152is oriented away from the prominent groove in the crystal structure, decreasing its direct accessibility to ssDNA bound at this site. Therefore to implicate Lys152 in binding ssDNA, the ssDNA would somehow have to wrap around the protein, suggesting that the interaction encompasses more residues than those lying directly within this groove. A structural based sequence alignment of other ssDNA-binding proteins does not reveal conservation at this particular residue (16Hollis T. Stattel J.M. Walther D.S. Richardson C.C. Ellenberger T. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 9557-9562Crossref PubMed Scopus (78) Google Scholar) and therefore, perhaps this interaction is a unique feature of the gene 2.5 protein.A second lethal mutation resulting in the alteration of a basic residue was identified at Lys109. In contrast to gp2.5-K152E, this K109I variant gene 2.5 protein failed to inhibit the ability of gene 2.5 protein to bind ssDNA despite the loss of a positively charged residue in the OB fold. Interestingly gp2.5-K109I displayed an aberrant binding pattern in the gel shift assay, forming only the slower mobility complex. This binding pattern was also exhibited by gp2.5-Δ26C, which likewise failed to form the rapidly migrating complex. Originally this binding pattern was thought to indicate a level of cooperative binding to ssDNA not characteristic of the native protein. However, upon closer examination, the appropriate kinetic calculations, i.e. Hill coefficients, could not support this theory. Further dissection of the binding mode(s) of the native protein may lead to an explanation of this observation.Based on the crystal structure a model was proposed for DNA binding that assumes that gene 2.5 binds ssDNA as a monomeric species (16Hollis T. Stattel J.M. Walther D.S. Richardson C.C. Ellenberger T. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 9557-9562Crossref PubMed Scopus (78) Google Scholar). The hypothesis is that the negatively charged, acidic carboxyl terminus competes with the proposed DNA binding site of an adjacent protomer leading to the formation of dimers in the absence of ssDNA. Therefore dissolution of the dimer would be necessary to expose the DNA binding domain and allow for ssDNA binding. In support of this model we have shown how a carboxyl-terminal truncated form of the protein gp 2.5-Δ26C that exists as a monomer in solution (35Rezende L.F. Hollis T. Ellenberger T. Richardson C.C. J. Biol. Chem. 2002; 277: 50643-50653Abstract Full Text Full Text PDF PubMed Scopus (22) Google Scholar) has a 10-fold greater affinity for ssDNA as compared with wild-type gene 2.5 protein. Similar results have been seen with gp2.5-Δ21C, also a monomer in solution, where ssDNA binding was analyzed by surface plasmon resonance. 4J. M. Stattel and C. C. Richardson, unpublished data. The electrophoretic mobility shift assay employed in this study provided an insight into the mode by which wild-type gene 2.5 protein binds ssDNA. Over a protein concentration series from 80 to 10,600 nm, upon binding a 70-mer oligonucleotide, two distinct protein·DNA complexes were resolved. Conceivably these two complexes could represent the binding of one monomer of gene 2.5 protein and a subsequent second monomer at a higher concentration. This interpretation is supported by the absence of the slower mobility complex when wild-type gene 2.5 protein binds to a shorter oligonucleotide of 38 bases in length as presumably only one monomer can be accommodated on this length. However, this hypothesis does not agree with the published site size for the protein, which is seven nucleotides per monomer (8Kim 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). This site size was calculated by assessing the binding of gene 2.5 protein to circular M13 ssDNA. In a subsequent study using surface plasmon resonance, stable binding of gene 2.5 protein required an oligonucleotide of at least 30 nucleotides in length.4 Nonetheless this unexplained phenomenon presented by the distinct protein·DNA complexes warrants further study especially because other ssDNA-binding proteins have demonstrated different modes of binding ssDNA.As gene 2.5 protein has the capacity to mediate homologous DNA annealing2 we examined how efficiently the altered proteins could accomplish this activity. The altered proteins deficient in binding to ssDNA were also defective in annealing complimentary strands of ssDNA. However, we do observe some annealing at higher protein concentrations. For this reason, we feel the lack of annealing we observed is related to the affinity of the altered proteins for ssDNA rather than reflecting a defect in the basic mechanism of homologous base-pairing. Interestingly, gp2.5-Δ26C facilitates strand annealing more efficiently than the wild-type protein,3 further supporting a relationship between ssDNA binding affinity and homologous base pair annealing. Furthermore, despite its unaltered affinity for ssDNA the variant protein K109I is defective in base pairing, requiring 2-fold more protein to completely anneal all the substrate. We are currently pursuing the mechanism of this reaction by studying another altered protein that binds ssDNA but cannot facilitate the annealing of homologous strands of ssDNA.3 From the data presented here, we conclude that gp2.5 must be able to bind ssDNA to facilitate DNA annealing.All of the proteins described in this study were expressed from lethal mutations in gene 2.5. It is likely that gp2.5-K152E, gp2.5-Y158C, and gp2.5-Y111C/Y158C are lethal because they have a lower affinity for ssDNA. Given that ssDNA binding is an essential function for gene 2.5 it is not surprising that amino acid changes that reduce binding affinity in vitro are lethal in vivo. In addition, we have shown that these proteins can still form dimers, and physically interact with the T7 DNA polymerase and the 63-kDa gene 4 protein. The mechanism underlying gp2.5-K109I lethality, on the other hand, remains unclear. In cells harboring a plasmid encoding for this genetic alteration, we detected a reduced level DNA synthesis in vivo upon infection by T7Δ2.5 bacteriophage, 5E. M. Hyland, L. F. Rezende, and C. C. Richardson, unpublished data. suggesting that the function of this residue is important in the overall DNA replication of the bacteriophage. It is conceivable that both the impaired ability to facilitate annealing and the altered manner in which it binds ssDNA accounts for the in vivo phenotype. However, further investigation is necessary to probe the precise molecular basis of its involvement in the life cycle of bacteriophage T7. Single-stranded DNA (ssDNA)1-binding proteins lack sequence specificity and bind ssDNA with a higher affinity than they bind double-stranded DNA or RNA (1Chase J.W. Williams K.R. Annu. Rev. Biochem. 1986; 55: 103-136Crossref PubMed Scopus (444) Google Scholar). Primarily, ssDNA-binding proteins function to bind any exposed regions of ssDNA in cells, forming a protective coat around the reactive bases and thus restricting the formation of secondary structures. However, their role is not restricted to extending and protecting DNA in that they also physically and functionally interact with other replication proteins. Bacteriophage T7 encodes its own ssDNA-binding protein, the product of gene 2.5. Gene 2.5 protein is essential for phage survival (2Kim Y.T. Richardson C.C. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 10173-10177Crossref PubMed Scopus (58) Google Scholar) and plays multiple roles in DNA replication, recombination, and repair (2Kim Y.T. Richardson C.C. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 10173-10177Crossref PubMed Scopus (58) Google Scholar, 3Reuben R.C. Gefter M.L. Proc. Natl. Acad. Sci. U. S. A. 1973; 70: 1846-1850Crossref PubMed Scopus (49) Google Scholar, 4Scherzinger E. Litfin F. Jost E. Mol. Gen. Genet. 1973; 123: 247-262Crossref PubMed Scopus (40) Google Scholar, 5Araki H. Ogawa H. Virology. 1981; 111: 509-515Crossref PubMed Scopus (18) Google Scholar, 6Araki H. Ogawa H. Mol. Gen. Genet. 1981; 183: 66-73Crossref PubMed Scopus (15) Google Scholar, 7Nakai H. Richardson C.C. J. Biol. Chem. 1988; 263: 9831-9839Abstract Full Text PDF PubMed Google Scholar, 8Kim 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, 9Kim Y.T. Tabor S. Churchich J.E. Richardson C.C. J. Biol. 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