Quantitative genomic analysis of RecA protein binding during DNA double-strand break repair reveals RecBCD action in vivo

Abstract : Understanding molecular mechanisms in the context of living cells requires the development of new methods of in vivo biochemical analysis to complement established in vitro biochemistry. A critically important molecular mechanism is genetic recombination, required for the beneficial reassortment of genetic information and for DNA double-strand break repair (DSBR). Central to recom-bination is the RecA (Rad51) protein that assembles into a spiral filament on DNA and mediates genetic exchange. Here we have developed a method that combines chromatin immunoprecipita-tion with next-generation sequencing (ChIP-Seq) and mathematical modeling to quantify RecA protein binding during the active repair of a single DSB in the chromosome of Escherichia coli. We have used quantitative genomic analysis to infer the key in vivo molecular parameters governing RecA loading by the helicase/ nuclease RecBCD at recombination hot-spots, known as Chi. Our genomic analysis has also revealed that DSBR at the lacZ locus causes a second RecBCD-mediated DSBR event to occur in the terminus region of the chromosome, over 1 Mb away. homologous recombination | mechanistic modelling | DNA repair | RecA |
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Charlotte A. Cockram, Milana Filatenkova, Vincent Danos, Meriem El Karoui, David R. F. Leach. Quantitative genomic analysis of RecA protein binding during DNA double-strand break repair reveals RecBCD action in vivo. Proceedings of the National Academy of Sciences of the United States of America , National Academy of Sciences, 2015, ⟨10.1073/pnas.1424269112⟩. ⟨hal-01263631⟩

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