Gene 4 helicase of bacteriophage T7 mediates strand transfer through pyrimidine dimers, mismatches, and nonhomologous regions

Abstract

In bacteriophage T7 the gene 2.5 singlestranded DNA-binding protein and the gene 4 helicase together promote the annealing of homologous regions of two DNA partners to form a joint molecule and subsequent strand transfer. In this reaction T7 gene 2.5 protein is essential for joint molecule formation, but is not required for T7 gene 4 protein-mediated strand transfer. T7 gene 4 helicase alone is able to mediate strand transfer, provided that a joint molecule is available. The present paper shows that, in addition, strand transfer proceeds at a normal rate even when both DNA partners contain ultraviolet-induced pyrimidine dimers (0.6 dimer per 100 nt). An insert of a relatively long (842-nt) segment of nonhomologous DNA in the single-stranded DNA partner has no effect on strand transfer, whereas its presence in the double-stranded partner prevents strand transfer. A short insert (37 nt) can be tolerated in either partner. Thus, DNA helicase is able to participate in recombinational DNA repair through its role in strand exchange, providing a pathway distinct from nucleotide excision repair.Recent studies have shown that the gene 41 DNA helicase encoded by bacteriophage T4 (1) and the gene 4 helicase encoded by bacteriophage T7 (2) mediate homologous DNA strand transfer within homologous DNA molecules. The rate and polarity of strand transfer are in accord with the properties of these enzymes in catalyzing strand displacement at a replication fork. A convenient DNA substrate for dissecting the strand transfer reaction is a joint DNA molecule formed between a single-stranded circle and a homologous linear duplex DNA molecule with a short 3Ј-single-stranded tail (Fig.

In Vitro Repair of Gaps in Bacteriophage T7 DNA

“…The RecBCD protein, a major enzymatic component of one of E. coli's recombination pathways, is inactivated by T7 soon after infection to allow the phage genome to escape devastation from RecBCD's exonuclease V activity (53). Recently it has been found that T7 single-strand-DNA binding protein and the helicase encoded by gene 4 play major roles in annealing complementary DNA strands together (16,19,20). Thus, formation of single-stranded DNA and annealing of those strands to form heteroduplexes may figure prominently in T7 recombination (42).…”

“…Bacteriophage T7 has provided a powerful tool with which to study DNA replication and offers several advantages as an experimental system. The genetics of T7 are well developed, and a considerable amount is known about the biochemistry of T7 enzymes involved in DNA metabolism (5,6,16,40,46). Efficient in vitro systems for DNA replication and DNA packaging have been developed (9,21,29).…”

Motors, Switches, and Contacts in the Replisome

“…Presumably these interactions explain why coordination of leading-and laggingstrand synthesis in vitro is dependent upon gene 2.5 protein (159). Furthermore, gp2.5 facilitates homologous DNA base pairing, a process that is important in the formation of T7 concatemers, in recombination (185)(186)(187), and in the repair of double-stranded breaks (188). The crystal structure of gp2.5 (189) shows that it has the conserved oligosaccharideoligonucleotide-binding fold (OB-fold) that is common to this group of proteins (190,191).…”

In Vitro Repair of Gaps in Bacteriophage T7 DNA

“…The RecBCD protein, a major enzymatic component of one of E. coli's recombination pathways, is inactivated by T7 soon after infection to allow the phage genome to escape devastation from RecBCD's exonuclease V activity (53). Recently it has been found that T7 single-strand-DNA binding protein and the helicase encoded by gene 4 play major roles in annealing complementary DNA strands together (16,19,20). Thus, formation of single-stranded DNA and annealing of those strands to form heteroduplexes may figure prominently in T7 recombination (42).…”