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 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.

authors

Cockram CA,Filatenkova M,Danos V,El Karoui M,Leach DR

doi

10.1073/pnas.1424269112

subject

Has Abstract

pub_date

2015-08-25 00:00:00

pages

E4735-42

issue

34

eissn

0027-8424

issn

1091-6490

pii

1424269112

journal_volume

112

pub_type

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