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J. Biol. Chem., Vol. 278, Issue 9, 7247-7256, February 28, 2003
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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 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-
26C, 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 gp2.5-26C mediates the reaction more effectively than
does wild-type. Gp2.5-K109I retains this annealing ability, albeit
slightly less efficiently. With the exception of gp2.5-26C, all
variant proteins form dimers in solution and physically 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.
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