Role of the bacteriophage T7 and 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 molecules 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 partners, a reaction that is mediated by the gene 2.5 protein alone. A mutant gene 2.5 protein, gene 2.5-Delta21C protein, lacking 21 amino acid 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 strand transfer by gene 4 helicase is inhibited by the presence of the gene 2.5-Delta21C protein. Bacteriophage T4 gene 32 protein likewise inhibits T7 gene 4 protein-mediated strand transfer, whereas Escherichia coli 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 synthesis of oligonucleotides at specific sequences during translocation on single-stranded DNA. We find that neither the rate nor extent of strand transfer is significantly affected by concurrent primer synthesis. The bacteriophage T4 gene 41 helicase has been shown to catalyze polar branch migration after the T4 gene 59 helicase assembly protein loads 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 partially single-stranded homologous DNA partners and that subsequent branch migration requires this single-stranded DNA-binding protein in addition to the gene 41 helicase and the gene 59 helicase assembly protein. Similar to the strand transfer reaction, strand displacement DNA synthesis catalyzed by T4 DNA polymerase also requires the presence of gene 32 protein in addition to the gene 41 and 59 proteins.
Kong
D
D
Department of Biological Chemistry and Molecular Pharmacology, Harvard Medical School, Boston, Massachusetts 02115, USA.
Nossal
N G
NG
Richardson
C C
CC
D017122:Bacteriophage T4; D017123:Bacteriophage T7; D004265:DNA Helicases / Q000378:metabolism*; D019915:DNA Primase; D004261:DNA Replication; D004277:DNA, Single-Stranded / Q000378:metabolism*; D004268:DNA-Binding Proteins / Q000378:metabolism*; D004587:Electrophoresis, Agar Gel; D008958:Models, Molecular; D012316:RNA Nucleotidyltransferases / Q000378:metabolism*; D014764:Viral Proteins / Q000378:metabolism*
D004277:DNA, Single-Stranded; D004268:DNA-Binding Proteins; D014764:Viral Proteins; C085913:gene 2.5 protein, Enterobacteria phage T7; C063983:gene 41 protein, Enterobacteria phage T4; C091225:gene 59 protein, Enterobacteria phage T4; C060035:gp32 protein, Enterobacteria phage T4; D019915:DNA Primase; D012316:RNA Nucleotidyltransferases; D004265:DNA Helicases