ROLE OF THE BACTERIOPHAGE-T7 AND BACTERIOPHAGE-T4 SINGLE-STRANDED DNA-BINDING PROTEINS IN THE FORMATION OF JOINT MOLECULES AND DNA HELICASE-CATALYZED POLAR BRANCH MIGRATION

Bacteriophage T7 gene 2.5 single-stranded DNA-binding protein and gene 4 DNA helicase together promote pairing of two homologous DNA molecul es and subsequent polar branch migration (Kong, D., and Richardson, C. C. (1996) EMBO J. 15, 2010-2019), In this report, we show that gene 2 .5 protein is not required for the initiation or propagation of strand transfer once a joint molecule has been formed between the two DNA pa rtners, a reaction that is mediated by the gene 2.5 protein alone. A m utant gene 2.5 protein, gene 2.5-Delta 21C protein, lacking 21 amino a cid residues at its C terminus, cannot physically interact with gene 4 protein, Although it does bind to single-stranded DNA and promote the formation of joint molecule via homologous base pairing, subsequent s trand transfer by gene 4 helicase is inhibited by the presence of the gene 2.5-Delta 21C protein, Bacteriophage T4 gene 32 protein likewise inhibits T7 gene 4 protein-mediated strand transfer, whereas Escherich ia coil single-stranded DNA-binding protein does not, The 63-kDa gene 4 protein of phage T7 is also a DNA primase in that it catalyzes the s ynthesis of oligonucleotides at specific sequences during translocatio n on single-stranded DNA, We find that neither the rate nor extent of strand transfer is significantly affected by concurrent primer synthes is, The bacteriophage T4 gene 41 helicase has been shown to catalyze p olar branch migration after the T4 gene 59 helicase assembly protein l oads the helicase onto joint molecules formed by the T4 UvsX and gene 32 proteins (Salinas, F., and Kodadek, T. (1995) Cell 82, 111-119). We find that gene 32 protein alone forms joint molecules between partial ly single-stranded homologous DNA partners and that subsequent branch migration requires this single-stranded DNA-binding protein in additio n to the gene 41 helicase and the gene 59 helicase assembly protein. S imilar to the strand transfer reaction, strand displacement DNA synthe sis catalyzed by T4 DNA polymerase also requires the presence of gene 32 protein in addition to the gene 41 and 59 proteins.