1. Quantitative genomic analysis of RecA protein binding during DNA double-strand break repair reveals RecBCD action in vivo.
- Author
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Cockram CA, Filatenkova M, Danos V, El Karoui M, and Leach DR
- Subjects
- Chromatin Immunoprecipitation, Rec A Recombinases genetics, DNA Damage, DNA Repair, Exodeoxyribonuclease V metabolism, Genome, Rec A Recombinases metabolism
- 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 recombination 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 immunoprecipitation 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.
- Published
- 2015
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