Abstract
Double-strand breaks pose a major threat to the genome and must be repaired accurately if structural and functional integrity are to be preserved. This is usually achieved via homologous recombination, which enables the ends of a broken DNA molecule to engage an intact duplex and prime synthesis of the DNA needed for repair. In Escherichia coli, repair relies on the RecBCD and RecA proteins, the combined ability of which to initiate recombination and form joint-molecule intermediates is well understood. To shed light on subsequent events, we exploited the I-SceI homing endonuclease of yeast to make breaks at I-SceI cleavage sites engineered into the chromosome. We show that survival depends on RecA and RecBCD, and that subsequent events can proceed via either of two pathways, one dependent on the RuvABC Holliday junction resolvase and the other on RecG helicase. Both pathways rely on PriA, presumably to facilitate DNA replication. We discuss the possibility that classical Holliday junctions may not be essential intermediates in repair and consider alternative pathways for RecG-dependent separation of joint molecules formed by RecA.
Publication types
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Research Support, Non-U.S. Gov't
MeSH terms
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Chromosomes, Bacterial / genetics
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Chromosomes, Bacterial / metabolism
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Colony Count, Microbial
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DNA Helicases / genetics
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DNA Helicases / metabolism*
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DNA Repair*
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DNA-Binding Proteins / genetics
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DNA-Binding Proteins / metabolism
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Deoxyribonucleases, Type II Site-Specific / metabolism
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Escherichia coli / genetics*
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Escherichia coli / growth & development
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Escherichia coli / metabolism
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Escherichia coli Proteins / genetics
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Escherichia coli Proteins / metabolism*
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Exodeoxyribonuclease V / genetics
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Exodeoxyribonuclease V / metabolism
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Genes, Bacterial
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Holliday Junction Resolvases / genetics
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Holliday Junction Resolvases / metabolism
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Integrases / genetics
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Integrases / metabolism
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Models, Molecular
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Mutation
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Rec A Recombinases / genetics
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Rec A Recombinases / metabolism
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Recombination, Genetic
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Replication Protein A
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Saccharomyces cerevisiae Proteins
Substances
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DNA-Binding Proteins
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Escherichia coli Proteins
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Replication Protein A
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Saccharomyces cerevisiae Proteins
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XerC protein, E coli
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RecG protein, E coli
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Integrases
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Rec A Recombinases
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Exodeoxyribonuclease V
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Holliday Junction Resolvases
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SCEI protein, S cerevisiae
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Deoxyribonucleases, Type II Site-Specific
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DNA Helicases