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J. Biol. Chem., Vol. 277, Issue 52, 50643-50653, December 27, 2002
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From the Department of Biological Chemistry and Molecular
Pharmacology, Harvard Medical School, Boston, Massachusetts 02115
Gene 2.5 of bacteriophage T7 is an essential gene
that encodes a single-stranded DNA-binding protein. T7 phage with gene
2.5 deleted can grow only on Escherichia coli cells that
express gene 2.5 from a plasmid. This complementation assay was used to
screen for lethal mutations in gene 2.5. By screening a library of
randomly mutated plasmids encoding gene 2.5, we identified 20 different single amino acid alterations in gene 2.5 protein that are lethal in vivo. The location of these essential residues within
the three-dimensional structure of gene 2.5 protein assists in the
identification of motifs in the protein. In this study we show that a
subset of these alterations defines the dimer interface of gene 2.5 protein predicted by the crystal structure. Recombinantly expressed and purified gene 2.5 protein-P22L, gene 2.5 protein-F31S, and gene 2.5 protein-G36S do not form dimers at salt concentrations where the
wild-type gene 2.5 protein exists as a dimer. The basis of the
lethality of these mutations in vivo is not known because altered proteins retain the ability to bind single-stranded DNA, anneal
complementary strands of DNA, and interact with T7 DNA polymerase.
To whom correspondence should be addressed: Dept. of Biological
Chemistry and Molecular Pharmacology, Harvard Medical School, 240 Longwood Ave., Boston, MA, 02115. Tel.: 617-432-1864; Fax: 617-432-3362; E-mail: ccr@hms.harvard.edu.
This article has been cited by other articles:
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  L. Shokri, B. Marintcheva, C. C. Richardson, I. Rouzina, and M. C. Williams Single Molecule Force Spectroscopy of Salt-dependent Bacteriophage T7 Gene 2.5 Protein Binding to Single-stranded DNA J. Biol. Chem., December 15, 2006; 281(50): 38689 - 38696. [Abstract] [Full Text] [PDF]
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B. Marintcheva, S. M. Hamdan, S.-J. Lee, and C. C. Richardson Essential Residues in the C Terminus of the Bacteriophage T7 Gene 2.5 Single-stranded DNA-binding Protein J. Biol. Chem., September 1, 2006; 281(35): 25831 - 25840. [Abstract] [Full Text] [PDF]
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 S. M. Hamdan, B. Marintcheva, T. Cook, S.-J. Lee, S. Tabor, and C. C. Richardson A unique loop in T7 DNA polymerase mediates the binding of helicase-primase, DNA binding protein, and processivity factor PNAS, April 5, 2005; 102(14): 5096 - 5101. [Abstract] [Full Text] [PDF]
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Z.-G. He and C. C. Richardson Effect of Single-stranded DNA-binding Proteins on the Helicase and Primase Activities of the Bacteriophage T7 Gene 4 Protein J. Biol. Chem., May 21, 2004; 279(21): 22190 - 22197. [Abstract] [Full Text] [PDF]
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Z.-G. He, L. F. Rezende, S. Willcox, J. D. Griffith, and C. C. Richardson The Carboxyl-terminal Domain of Bacteriophage T7 Single-stranded DNA-binding Protein Modulates DNA Binding and Interaction with T7 DNA Polymerase J. Biol. Chem., August 8, 2003; 278(32): 29538 - 29545. [Abstract] [Full Text] [PDF]
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L. F. Rezende, S. Willcox, J. D. Griffith, and C. C. Richardson A Single-stranded DNA-binding Protein of Bacteriophage T7 Defective in DNA Annealing J. Biol. Chem., August 1, 2003; 278(31): 29098 - 29105. [Abstract] [Full Text] [PDF]
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E. M. Hyland, L. F. Rezende, and C. C. Richardson The DNA Binding Domain of the Gene 2.5 Single-stranded DNA-binding Protein of Bacteriophage T7 J. Biol. Chem., February 21, 2003; 278(9): 7247 - 7256. [Abstract] [Full Text] [PDF]
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