T7 Single Strand DNA Binding Protein but Not T7 Helicase Is Required for DNA Double Strand Break Repair

Abstract

An in vitro system based on Escherichia coli infected with bacteriophage T7 was used to test for involvement of host and phage recombination proteins in the repair of double strand breaks in the T7 genome. Double strand breaks were placed in a unique XhoI site located approximately 17% from the left end of the T7 genome. In one assay, repair of these breaks was followed by packaging DNA recovered from repair reactions and determining the yield of infective phage. In a second assay, the product of the reactions was visualized after electrophoresis to estimate the extent to which the double strand breaks had been closed. Earlier work demonstrated that in this system double strand break repair takes place via incorporation of a patch of DNA into a gap formed at the break site. In the present study, it was found that extracts prepared from uninfected E. coli were unable to repair broken T7 genomes in this in vitro system, thus implying that phage rather than host enzymes are the primary participants in the predominant repair mechanism. Extracts prepared from an E. coli recA mutant were as capable of double strand break repair as extracts from a wild-type host, arguing that the E. coli recombinase is not essential to the recombinational events required for double strand break repair. In T7 strand exchange during recombination is mediated by the combined action of the helicase encoded by gene 4 and the annealing function of the gene 2.5 single strand binding protein. Although a deficiency in the gene 2.5 protein blocked double strand break repair, a gene 4 deficiency had no effect. This argues that a strand transfer step is not required during recombinational repair of double strand breaks in T7 but that the ability of the gene 2.5 protein to facilitate annealing of complementary single strands of DNA is critical to repair of double strand breaks in T7.Double strand breaks are a frequent hazard to DNA molecules. These breaks can result from a number of causes, including DNA damage, fractures at progressing replication forks, or as part of normal homologous recombination (10,15,26,37,61). Failure to repair a double strand break can be lethal. Improper repair of double strand breaks can lead to deletions or chromosome aberrations (9, 12, 52). To minimize the deleterious effects of double strand breaks, living organisms maintain an array of repair mechanisms able to correct double strand breaks or rescue partial genomes formed as a result of breaks (3,10,12,21,22,26,38,39). Recombinationinduced DNA replication is a major route to rescue of partial genomes formed by collapse of replication forks in prokaryotes (26). In Escherichia coli, stable DNA replication is induced by forming new replication forks at sites other than the normal origin of replication (22,27,32). In bacteriophage T4, recombination between partial genomes formed by double strand breaks and intact T4 DNA molecules induces new replication forks that comprise a major part of the phage's DNA replication cycle (25, 36). It has long been appreciated that reco...

Single Molecule Force Spectroscopy of Salt-dependent Bacteriophage T7 Gene 2.5 Protein Binding to Single-stranded DNA

“…It physically interacts with both T7 DNA polymerase and the T7 helicase/primase (22)(23)(24)(25)(26) and plays multiple roles in T7 DNA replication and recombination (22,(27)(28)(29)(30)(31)(32)(33)(34)(35)(36). In the absence of DNA, gp2.5 forms a stable homodimer in solution (35).…”

The DNA Binding Domain of the Gene 2.5 Single-stranded DNA-binding Protein of Bacteriophage T7

“…Presumably these interactions explain why coordination of leading and lagging strand synthesis in vitro is dependent upon gene 2.5 protein (13). Furthermore, gene 2.5 protein facilitates homologous DNA base pairing, 2 a process that is important during viral recombination (10,11,14) and in the repair of doublestranded breaks in the T7 chromosome (12).…”

“…Bacteriophage T7 encodes its own ssDNAbinding protein, the product of gene 2.5. Gene 2.5 protein is essential for phage survival (2) and plays multiple roles in DNA replication, recombination, and repair (2)(3)(4)(5)(6)(7)(8)(9)(10)(11)(12). Gene 2.5 protein interacts directly with both the T7 DNA polymerase (9) and the gene 4 helicase/primase (7), stimulating the activity of each protein.…”

Essential Amino Acid Residues in the Single-stranded DNA-binding Protein of Bacteriophage T7

“…It encodes a single-stranded DNA (ssDNA) 1 -binding protein that is functionally similar to the Escherichia coli SSB protein and the gene 32 protein of bacteriophage T4 (2,3). Like these ssDNA-binding proteins, the gene 2.5 product (wt gene 2.5 protein) is important for DNA replication, recombination, and repair (1)(2)(3)(4)(5)(6)(7)(8)(9)(10)(11)(12). However, neither the E. coli SSB protein nor the T4 gene 32 protein can replace gene 2.5 protein in cells infected by T7 phage lacking gene 2.5 (13).…”