Repair of double‐strand breaks by incorporation of a molecule of homologous DNA

Abstract

SummaryAn in vitro system based upon extracts of Escherichia coli infected with bacteriophage T7 was used to monitor repair of double-strand breaks in the T7 genome. The efficiency of double-strand break repair was markedly increased by DNA molecules (`donor' DNA) consisting of a 2.1 kb DNA fragment, generated by PCR, that had ends extending < 1 kb on either side of the break site. Repair proceeded with greater than 10% efficiency even when T7 DNA replication was inhibited. When the donor DNA molecules were labelled with 32 P, repaired genomes incorporated label only near the site of the double-strand break. When repair was carried out with unlabelled donor DNA and [ 32 P]-dCTP provided as precursor for DNA synthesis the small amount of incorporated label was distributed randomly throughout the entire T7 genome. Repair was performed using donor DNA that had adjacent BamHI and PstI sites. When the BamHI site was methylated and the PstI site was left unmethylated, the repaired genomes were sensitive to PstI but not to BamHI endonuclease, showing that the methyl groups at the BamHI recognition site had not been replaced by new DNA synthesis during repair of the double-strand break. These observations are most consistent with a model for double-strand break repair in which the break is widened to a small gap, which is subsequently repaired by physical incorporation of a patch of donor DNA into the gap.

A T3 and T7 Recombinant Phage Acquires Efficient Adsorption and a Broader Host Range

“…The mechanism of recombination between two T7 group's phages remains unclear. Repair of the T7 double strand breakage (DSB) using a 2.1 kb PCR fragment as a donor resulted in the incorporation of a patch of the donor in vitro, suggesting a strand annealing model of repair [13]. However, a short fragment is especially susceptible to degradation and unwinding, while how a requisite repair patch will be produced for the full-length genome donor is not known.…”

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

“…These reactions (data not shown) did not show extensive degradation of unused donor DNA or unrepaired partial genomes. However, this type of DNA degradation was apparent in our earlier experiments which had been performed using phage that had normal levels of gene 2.5 and gene 6 proteins (28,29). Since the undigested BstXI fragments obscured detection of the KpnI D fragment that is diagnostic of complete repair, 2.1-kb PCR fragments extending from positions 5594 to 7729 were used in place of the BstXI fragments as donor DNA.…”

“…Under conditions where the major DNA replication pathway is blocked, T7 repairs double strand breaks by inserting fragments of donor DNA into a gap created at the break site (29). Recombination in the region of overlap between the donor DNA and the partial genomes created by the double strand break could be mediated by either E. coli or T7 proteins .…”

“…The reaction mixtures were extracted with phenol-chloroform and precipitated with ethanol, and the DNA was resuspended in 1/10 volume Tris-EDTA (10 mM Tris-HCl [pH 7.5], 0.1 mM EDTA). The DNA was treated with boiled bovine pancreas RNase a (final concentration, 1 mg/ml, Calbiochem) and digested with KpnI, and 0.014 ml was subjected to electrophoresis at 24 V for 4 h in a 0.4% agarose gel in Tris acetate-EDTA buffer (28,29). The gel were stained with ethidium bromide to give the patterns shown in the figures.…”

“…Under conditions where DNA replication is blocked, double strand breaks in a T7 genome are repaired by insertion of a patch of donor DNA into a gap formed at the break site (29). The insertion process provides a very efficient repair mechanism, sealing about half of the double strand breaks even under conditions where every genome in the reactions has a double strand break.…”