Mechanism of Annealing of Complementary DNA Strands by the Single-Stranded DNA Binding Protein of Bacteriophage T7

Abstract

Gp2.5, an essential single-stranded DNA-binding protein encoded by bacteriophage T7, is integral to various steps of DNA metabolism. Unlike other single-stranded DNA binding proteins, it greatly facilitates the annealing of complementary DNA strands. Gp2.5 efficiently anneals DNA duplexes as short as 30 base pairs: efficient annealing occurs at a 100-fold lower concentration of complementary strands than that required in the absence of gp2.5. Additionally, gp2.5 selectively promotes DNA annealing with no observed effect on RNA or DNA hybrids. Kinetic studies show a substantial increase in the annealing rate, with gp2.5 accelerating the process by 30-fold compared with spontaneous annealing. Gp2.5 tolerates mismatches and unpaired loops within DNA, facilitating annealing in sequences with slight imperfections. FRET analysis demonstrates that gp2.5 brings strands of ssDNA into close proximity irrespective of their complementarity, likely through interactions between gp2.5 molecules. A unique long α helix A in gp2.5 is critical for its annealing activity: deletions of helix A impair DNA annealing without affecting DNA replication functions.