Details of the nucleic acid binding site of T4 gene 32 protein revealed by proteolysis and DNA T m depression methods 1 1Edited by R. Ebright

Wu, Min; Flynn, Elizabeth K.; Karpel, Richard L.

doi:10.1006/jmbi.1999.2541

Cited by 18 publications

(29 citation statements)

References 25 publications

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“…Expression was induced with 0.1 mg/ml nalidixic acid when the cells reached an A 595 of 0.8 -0.9, and the proteins were isolated on denatured DNA-cellulose as described previously (7,17,18). The chromatographic properties of *I were similar to that of whole protein; peak elution was at ϳ0.8 M NaCl.…”

Section: Methodsmentioning

confidence: 99%

“…showed that although the core domain of gene 32 protein was susceptible to the action of mammalian endoproteinase Arg-C, the intact protein was refractory to digestion (7,9). To determine whether the presence of the N-or the C-terminal domain is responsible for this protection, we compared the endo Arg-C digestion behavior of all three truncated products.…”

Section: The Presence Of the C-terminal Domain Reduces The Susceptibimentioning

confidence: 99%

“…We have observed that the residues within the N-terminal domain most essential for homotypic protein-protein interaction, Lys (3)-Arg (4)-Lys (5)-Ser (6)-Thr (7), are homologous to the aforementioned LAST residues within the core domain, Lys (110)-Arg (111)-Lys (112)-Thr (113)-Ser (114). On the basis of this and other observations, we devised a model for the cooperative binding of 32 protein (8,9).…”

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confidence: 99%

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Domain Effects on the DNA-interactive Properties of Bacteriophage T4 Gene 32 Protein

Waidner

Flynn

et al. 2001

Journal of Biological Chemistry

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Bacteriophage T4 gene 32 protein, a model for singlestrand specific nucleic acid-binding proteins, consists of three structurally and functionally distinct domains. We have studied the effects of the N and C domains on the protein structure and its nucleic acid-interactive properties. Although the presence of the C domain decreases the proteolytic susceptibility of the core (central) domain, quenching of the core tryptophan fluorescence by iodide is unaltered by the presence of the terminal domains. These results suggest that the overall conformation of the core domain remains largely independent of the flanking domains. Removal of the N or the C terminus does not abolish the DNA renaturation activity of the protein. However, intact protein and its three truncated forms differ in DNA helix-destabilizing activity. The C domain alone is responsible for the kinetic barrier to natural DNA helix destabilization seen with intact protein. Intact protein and core domain potentiate the DNA helix-destabilizing activity of truncated protein lacking only the C domain (*I), enhancing the observed hyperchromicity while increasing the melting temperature. Proteolysis experiments suggest that the affinity of core domain for single-stranded DNA is increased in the presence of *I. We propose that *I can "mingle" with intact protein or core domain while bound to single-stranded DNA.

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Section: Methodsmentioning

confidence: 99%

Section: The Presence Of the C-terminal Domain Reduces The Susceptibimentioning

confidence: 99%

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confidence: 99%

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Domain Effects on the DNA-interactive Properties of Bacteriophage T4 Gene 32 Protein

Waidner

Flynn

et al. 2001

Journal of Biological Chemistry

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“…The higher affinity of gp2.5-F232L for ssDNA is thus compatible with the model on the basis that the single amino acid change in the tail decreases its affinity for the DNA-binding site of the protein. It has been proposed that the interaction of the carboxyl tail of the T4 gene 32 ssDNA-binding protein with its DNA-binding site modulates its affinity for ssDNA (49). Gene 2.5 protein is a dimer in solution (7) and in the crystal structure (26).…”

Section: Figmentioning

confidence: 99%

The Carboxyl-terminal Domain of Bacteriophage T7 Single-stranded DNA-binding Protein Modulates DNA Binding and Interaction with T7 DNA Polymerase

Rezende

Willcox

et al. 2003

Journal of Biological Chemistry

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Gene 2.5 of bacteriophage T7 is an essential gene that encodes a single-stranded DNA-binding protein (gp2.5). Previous studies have demonstrated that the acidic carboxyl terminus of the protein is essential and that it mediates multiple protein-protein interactions. A screen for lethal mutations in gene 2.5 uncovered a variety of essential amino acids, among which was a single amino acid substitution, F232L, at the carboxyl-terminal residue. gp2.5-F232L exhibits a 3-fold increase in binding affinity for single-stranded DNA and a slightly lower affinity for T7 DNA polymerase when compared with wild type gp2.5. gp2.5-F232L stimulates the activity of T7 DNA polymerase and, in contrast to wild-type gp2.5, promotes strand displacement DNA synthesis by T7 DNA polymerase. A carboxyl-terminal truncation of gene 2.5 protein, gp2.5-⌬26C, binds single-stranded DNA 40-fold more tightly than the wild-type protein and cannot physically interact with T7 DNA polymerase. gp2.5-⌬26C is inhibitory for DNA synthesis catalyzed by T7 DNA polymerase on single-stranded DNA, and it does not stimulate strand displacement DNA synthesis at high concentration. The biochemical and genetic data support a model in which the carboxyl-terminal tail modulates DNA binding and mediates essential interactions with T7 DNA polymerase.Gene 2.5 of bacteriophage T7 encodes a single-stranded DNA (ssDNA) 1 -binding protein (gp2.5) that is essential for viral survival (1). gp2.5 modulates several important reactions in DNA replication, recombination, and repair (1-12). The fundamental reactions at the T7 phage replication fork can be reconstituted with only four proteins (13, 14): T7 gene 5 DNA polymerase, its processivity factor Escherichia coli thioredoxin (15, 16), T7 gene 4 helicase/primase (17-19), and T7 gp2.5. gp2.5 physically interacts with T7 DNA polymerase and T7 helicase/primase to stimulate their activities (6,8). The binding of gp2.5 to ssDNA is critical because it affects both specific DNA-protein and protein-protein interactions in the replisome (14,20). In this regard it is essential for coupling leading and lagging strand DNA synthesis in vitro (14). gp2.5 is also essential for recombination in T7 phage-infected cells, and in addition to the interactions described above, it also mediates homologous base pairing (11).Despite a lack of sequence homology, T7 gp2.5 is functionally similar to the extensively studied SSB protein of E. coli and the gene 32 protein of bacteriophage T4. Like gp2.5, they are both ssDNA-binding proteins, a class of ubiquitous proteins that are not only essential in DNA replication but also play key roles in DNA recombination and repair (7, 21). Biochemical studies have shown that these proteins, like T7 gp2.5, interact with other proteins at the replication fork. E. coli SSB protein interacts with E. coli DNA polymerase II, exonuclease I, and other proteins involved in replication (22-24). T4 gene 32 protein physically interacts with at least 10 T4-encoded proteins, including T4 DNA polymerase, that are involved in T4...

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“…Interactions of the C-terminal tail with the DNA binding site of gp2.5 could modulate its affinity for ssDNA, as was proposed for the acidic C-terminal tail of the T4 gene 32 single-stranded binding protein (40). The binding of ssDNA might act as a switch that displaces the tail so it is free to interact with the other proteins of the T7 replication complex.…”

Section: Figmentioning

confidence: 99%

Structure of the gene 2.5 protein, a single-stranded DNA binding protein encoded by bacteriophage T7

Hollis

Stattel

Walther

et al. 2001

Proc. Natl. Acad. Sci. U.S.A.

118

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The gene 2.5 protein (gp2.5) of bacteriophage T7 is a singlestranded DNA (ssDNA) binding protein that has essential roles in DNA replication and recombination. In addition to binding DNA, gp2.5 physically interacts with T7 DNA polymerase and T7 primasehelicase during replication to coordinate events at the replication fork. We have determined a 1.9-Å crystal structure of gp2.5 and show that it has a conserved OB-fold (oligosaccharide͞oligonucle-otide binding fold) that is well adapted for interactions with ssDNA. Superposition of the OB-folds of gp2.5 and other ssDNA binding proteins reveals a conserved patch of aromatic residues that stack against the bases of ssDNA in the other crystal structures, suggesting that gp2.5 binds to ssDNA in a similar manner. An acidic C-terminal extension of the gp2.5 protein, which is required for dimer formation and for interactions with the T7 DNA polymerase and the primase-helicase, appears to be flexible and may act as a switch that modulates the DNA binding affinity of gp2.5.

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Details of the nucleic acid binding site of T4 gene 32 protein revealed by proteolysis and DNA T m depression methods 1 1Edited by R. Ebright

Cited by 18 publications

References 25 publications

Domain Effects on the DNA-interactive Properties of Bacteriophage T4 Gene 32 Protein

Domain Effects on the DNA-interactive Properties of Bacteriophage T4 Gene 32 Protein

The Carboxyl-terminal Domain of Bacteriophage T7 Single-stranded DNA-binding Protein Modulates DNA Binding and Interaction with T7 DNA Polymerase

Structure of the gene 2.5 protein, a single-stranded DNA binding protein encoded by bacteriophage T7

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