Double-Strand Break Repair in Bacteriophage T4: Recombination Effects of 3′–5′ Exonuclease Mutations

Abstract

The role of 39-59 exonucleases in double-strand break (DSB)-promoted recombination was studied in crosses of bacteriophage T4, in which DSBs were induced site specifically within the rIIB gene by SegC endonuclease in the DNA of only one of the parents. Frequency of rII 1 recombinants was measured in two-factor crosses of the type i 3 ets1, where ets1 designates an insertion in the rIIB gene carrying the cleavage site for SegC and i's are rIIB or rIIA point mutations located at various distances (12-2040 bp) from the ets1 site. The frequency/distance relationship was obtained in crosses of the wild-type phage and dexA1 (deficiency in deoxyribonuclease A), D219A (deficiency in the proofreading exonuclease of DNA polymerase), and tsL42 (antimutator allele of DNA polymerase) mutants. In all the mutants, recombinant frequency in crosses with the i-markers located at 12 and 33 bp from ets1 was significantly enhanced, implying better preservation of 39-terminal sequences at the ends of the broken DNA. The effects of dexA1 and D219A were additive, suggesting an independent action of the corresponding nucleases in the DSB repair pathway. The recombination enhancement in the dexA1 mutant was limited to short distances (,100 bp from ets1), whereas in the D219A mutant a significant enhancement was seen at all the tested distances. From the character of the frequency/distance relationship, it is inferred that the synthesisdependent strand-annealing pathway may operate in the D219A mutant. The recombination-enhancing effect of the tsL42 mutation could be explained by the hypothesis that the antimutator 43Exo removes a shorter stretch of paired nucleotides than the wild-type enzyme does during hydrolysis of the unpaired terminus in the D-loop intermediate. The role of the proofreading exonuclease in the formation of a robust replicative fork is discussed. S HCHERBAKOV et al. (2002) developed a modelsystem for studying double-strand break (DSB) repair based on the ets1 segCD strain of bacteriophage T4. A 66-bp fragment of phage T2L containing the cleavage site for SegC endonuclease (ets1) was inserted into the proximal part of the rIIB gene in a T4 segCD strain. In crosses of the ets1 segCD with a segC 1 partner, SegC endonuclease makes a DSB in the middle of the ets1 insertion, thus promoting recombination on both sides of ets1. This focused recombination is an effective tool for studying genetic recombination and DSB repair in vivo.The splice/patch coupling (SPC) pathway, substantiated earlier for the general genetic recombination and recombination-dependent replication (RDR) in bacteriophage T4 (Shcherbakov et al. 1992), and corroborated also for the DSB-promoted recombination (Shcherbakov et al.

Coordination and Processing of DNA Ends During Double-Strand Break Repair: The Role of the Bacteriophage T4 Mre11/Rad50 (MR) Complex

“…Thus, the marker that is 15 bp from the insertion is actually 68 bp from the 39 end of the broken DNA molecule. Regarding the resection events on the broken DNA, Shcherbakov et al (2006a) showed that the T4 exonuclease DexA contributes to erosion of DNA only very near a break and that the T4 DNA polymerase proofreading exonuclease contributes to erosion of DNA both near and farther from a break (both tested in an MR-proficient infection). The simplest model to explain our results is that the reduced amount of MR complex in the suppressed triple amber mutant allows increased access to DexA exonuclease (given the strong effect that we see with the nearby marker FC11) and perhaps also to the T4 DNA polymerase exonuclease.…”

“…In our previous study of 46 + infections, we found that the co-conversion frequency is very high near the break but drops off as a function of distance from the DSB (Stohr and Kreuzer 2002). We argued that exonuclease action starting at the break usually destroys the wild-type sequence on the cut chromosome when the flanking marker is very close to the break, but that the exonuclease action decreases stochastically with distance from the break (Stohr and Kreuzer 2002; also see Shcherbakov et al 2006a).…”