Author:
Cockram Charlotte A.,Filatenkova Milana,Danos Vincent,El Karoui Meriem,Leach David R. F.
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 ofEscherichia 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 thelacZlocus causes a second RecBCD-mediated DSBR event to occur in the terminus region of the chromosome, over 1 Mb away.
Funder
Medical Research Council
Marie Curie Cancer Care
EC | European Research Council
Publisher
Proceedings of the National Academy of Sciences
Cited by
45 articles.
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