Quantitative genomic analysis of RecA protein binding during DNA double-strand break repair reveals RecBCD action in vivo.
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.
Institute of Cell Biology, School of Biological Sciences, University of Edinburgh, Edinburgh EH9 3BF, United Kingdom;
Charlotte A
CA
Cockram
Life Sciences Institute, School of Informatics, University of Edinburgh, Edinburgh EH8 9LE, United Kingdom; SynthSys, Centre for Synthetic and Systems Biology, University of Edinburgh, Edinburgh EH9 3BF, United Kingdom.
Milana
M
Filatenkova
Life Sciences Institute, School of Informatics, University of Edinburgh, Edinburgh EH8 9LE, United Kingdom; SynthSys, Centre for Synthetic and Systems Biology, University of Edinburgh, Edinburgh EH9 3BF, United Kingdom.
Vincent
V
Danos
Institute of Cell Biology, School of Biological Sciences, University of Edinburgh, Edinburgh EH9 3BF, United Kingdom; SynthSys, Centre for Synthetic and Systems Biology, University of Edinburgh, Edinburgh EH9 3BF, United Kingdom Meriem.Elkaroui@ed.ac.uk D.Leach@ed.ac.uk.
Meriem
M
El Karoui
Institute of Cell Biology, School of Biological Sciences, University of Edinburgh, Edinburgh EH9 3BF, United Kingdom; Meriem.Elkaroui@ed.ac.uk D.Leach@ed.ac.uk.
David R F
DR
Leach
D047369:Chromatin Immunoprecipitation; D004249:DNA Damage; D004260:DNA Repair; D043211:Exodeoxyribonuclease V / Q000378:metabolism*; D016678:Genome; D011938:Rec A Recombinases / Q000235:genetics / Q000378:metabolism*
D011938:Rec A Recombinases; D043211:Exodeoxyribonuclease V
DNA repair; RecA; RecBCD; homologous recombination; mechanistic modelling