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Papers In Press, published online ahead of print October 12, 2002
J. Biol. Chem, 10.1074/jbc.M207359200
Submitted on July 22, 2002
Revised on October 11, 2002
Accepted on October 12, 2002
Department of Biological Chemistry and Molecular Pharmacology, Harvard Medical School, Boston, MA 02115
Corresponding Author: ccr{at}hms.harvard.edu
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 twenty 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 since altered proteins retain the ability to bind single-stranded DNA, anneal complementary strands of DNA, and interact with T7 DNA polymerase.
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