RESUMO
DNA double-strand break repair can be accomplished by homologous recombination when a sister chromatid or a homologous chromosome is available. However, 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 two essentially identical DNA sequences. We have developed a system using the Escherichia coli chromosome and the restriction enzyme EcoKI, in which double-strand breaks can be introduced into only one sister chromatid. We have shown that the components of the RecBCD and RecFOR 'pathways' are required for the recombinational repair of these breaks. Furthermore, we have shown a requirement for SbcCD, the prokaryotic homologue of Rad50/Mre11. This is the first demonstration that, like Rad50/Mre11, SbcCD is required for recombination in a wild-type cell. Our work suggests that the SbcCD-Rad50/Mre11 family of proteins, which have two globular domains separated by a long coiled-coil linker, is specifically required for the co-ordination of double-strand break repair reactions in which two DNA ends are required to recombine at one target site.
Assuntos
Cromossomos Bacterianos/metabolismo , Dano ao DNA , Reparo do DNA , Enzimas de Restrição do DNA/metabolismo , Proteínas de Escherichia coli , Escherichia coli/genética , Recombinação Genética , 2-Aminopurina/farmacologia , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Quebra Cromossômica/genética , Cromossomos Bacterianos/genética , Dano ao DNA/efeitos dos fármacos , Replicação do DNA , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Desoxirribonucleases/genética , Desoxirribonucleases/metabolismo , Escherichia coli/enzimologia , Exonucleases/genética , Exonucleases/metabolismo , Modelos Genéticos , MutaçãoRESUMO
The recombination mechanisms that deal with double-strand breaks in organisms as diverse as phage, bacteria, yeast, and humans are remarkably conserved. We discuss conservation in the biochemical pathways required to recombine DNA ends and in the structure of the DNA products. In addition, we highlight that two fundamentally distinct broken DNA substrates exist and describe how they are repaired differently by recombination. Finally, we discuss the need to coordinate recombinational repair with cell division through DNA damage response pathways.
Assuntos
Dano ao DNA/genética , Reparo do DNA/genética , DNA/química , DNA/metabolismo , Recombinação Genética/genética , Animais , Bacteriófago T4/genética , DNA/genética , Replicação do DNA , Escherichia coli/genética , Células Eucarióticas/metabolismo , Evolução Molecular , Humanos , Modelos Genéticos , Homologia de Sequência do Ácido NucleicoRESUMO
The Holliday junction is a central intermediate in homologous recombination. It consists of a four-way structure that can be resolved by cleavage to give either the crossover or noncrossover products observed. We show here that the formation of these products is controlled by the E. coli resolvasome (RuvABC) in such way that double-strand break repair (DSBR) leads to crossing over and single-strand gap repair (SSGR) does not lead to crossing over. We argue that the positioning of the RuvABC complex and its consequent direction of junction-cleavage is not random. In fact, the action of the RuvABC complex avoids crossing over in the most commonly predicted situations where Holliday junctions are encountered in DNA replication and repair. Our observations suggest that the positioning of the resolvasome may provide a general biochemical mechanism by which cells can control crossing over in recombination.
Assuntos
Troca Genética , Reparo do DNA , Endodesoxirribonucleases/metabolismo , Proteínas de Escherichia coli , Escherichia coli/genética , Proteínas de Bactérias/metabolismo , Dano ao DNA , DNA Helicases/metabolismo , Proteínas de Ligação a DNA/metabolismo , Escherichia coli/enzimologia , Escherichia coli/efeitos da radiação , Genótipo , Resolvases de Junção Holliday , Modelos Genéticos , Mutagênese , Transdução Genética , Raios UltravioletaRESUMO
A 246-bp imperfect palindrome has the potential to form hairpin structures in single-stranded DNA during replication. Genetic evidence suggests that these structures are converted to double-strand breaks by the SbcCD nuclease and that the double-strand breaks are repaired by recombination. We investigated the role of a range of recombination mutations on the viability of cells containing this palindrome. The palindrome was introduced into the Escherichia coli chromosome by phage lambda lysogenization. This was done in both wt and sbcC backgrounds. Repair of the SbcCD-induced double-strand breaks requires a large number of proteins, including the components of both the RecB and RecF pathways. Repair does not involve PriA-dependent replication fork restart, which suggests that the double-strand break occurs after the replication fork has passed the palindrome. In the absence of SbcCD, recombination still occurs, probably using a gap substrate. This process is also PriA independent, suggesting that there is no collapse of the replication fork. In the absence of RecA, the RecQ helicase is required for palindrome viability in a sbcC mutant, suggesting that a helicase-dependent pathway exists to allow replicative bypass of secondary structures.