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

Charlotte A. Cockram (1) , Milana Filatenkova (2) , Vincent Danos (3, 2) , Meriem El Karoui (1) , David R. F. Leach (1)

1 Edin. - University of Edinburgh
2 School of Informatics [Edimbourg]
3 ANTIQUE - Analyse Statique par Interprétation Abstraite

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 |

Domaines: Informatique [cs]; Sciences du Vivant [q-bio]

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, 2015, ⟨10.1073/pnas.1424269112⟩. ⟨hal-01263631⟩