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

Charlotte A. Cockram, Milana Filatenkova, Vincent Danos, Meriem El Karoui, David R F Leach

Research output: Contribution to journalArticlepeer-review

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.

Original languageEnglish
Article numberE4735-E4742
JournalProceedings of the National Academy of Sciences
Volume112
Issue number34
DOIs
Publication statusPublished - 25 Aug 2015

Keywords

  • DNA repair
  • Homologous recombination
  • Mechanistic modelling
  • RecA
  • RecBCD

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