Recombinational repair of chromosomal DNA double-strand breaks generated by a restriction endonuclease

DNA double-strand break repair can be accomplished by homologous recombinat ion when a sister chromatid or a homologous chromosome is available. Howeve r, the study of sister chromatid double-strand break repair in prokaryotes is complicated by the difficulty in targeting a break to only one copy of t wo essentially identical DNA sequences. We have developed a system using th e Escherichia coli chromosome and the restriction enzyme EcoKI, in which do uble-strand breaks can be introduced into only one sister chromatid. We hav e shown that the components of the RecBCD and RecFOR 'pathways' are require d for the recombinational repair of these breaks. Furthermore, we have show n a requirement for SbcCD, the prokaryotic homologue of Rad50/Mre11. This i s the first demonstration that, like Rad50/Mre11, SbcCD is required for rec ombination in a wild-type cell. Our work suggests that the SbcCD-Rad50/Mre1 1 family of proteins, which have two globular domains separated by a long c oiled-coil linker, is specifically required for the co-ordination of double -strand break repair reactions in which two DNA ends are required to recomb ine at one target site.