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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.

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