Role of the acidic carboxyl-terminal domain of the single-stranded DNA-binding protein of bacteriophage T7 in specific protein-protein interactions.

Abstract:

The gene 2.5 single-stranded DNA (ssDNA) binding protein of bacteriophage T7 is essential for T7 DNA replication and recombination. Earlier studies have shown that the COOH-terminal 21 amino acids of the gene 2.5 protein are essential for specific protein-protein interaction with T7 DNA polymerase and T7 DNA helicase/primase. A truncated gene 2.5 protein, in which the acidic COOH-terminal 21 amino acid residues are deleted no longer supports T7 growth, forms dimers, or interacts with either T7 DNA polymerase or T7 helicase/primase in vitro. The single-stranded DNA-binding protein encoded by Escherichia coli (SSB protein) and phage T4 (gene 32 protein) also have acidic COOH-terminal domains, but neither protein can substitute for T7 gene 2.5 protein in vivo. To determine if the specificity for the protein-protein interaction involving gene 2.5 protein resides in its COOH terminus, we replaced the COOH-terminal region of the gene 2.5 protein with the COOH-terminal region from either E. coli SSB protein or T4 gene 32 protein. Both of the two chimeric proteins can substitute for T7 gene 2.5 protein to support the growth of phage T7. The two chimeric proteins, like gene 2.5 protein, form dimers and interact with T7 DNA polymerase and helicase/primase to stimulate their activities. In contrast, chimeric proteins in which the COOH terminus of T7 gene 2.5 protein replaced the COOH terminus of E. coli SSB protein or T4 gene 32 protein cannot support the growth of phage T7. We conclude that an acidic COOH terminus of the gene 2.5 protein is essential for protein-protein interaction, but it alone cannot account for the specificity of the interaction.

Polymerases:

T7

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