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.homologous recombination | mechanistic modelling | DNA repair | RecA | RecBCD

Division-induced DNA double strand breaks in the chromosome terminus region of Escherichia coli lacking RecBCD DNA repair enzyme

“…As previously reported [51] we detected a weak increase of RecA ChIP downstream of Chi when cells were grown in LB, but we did not detect any increase in cells grown in M9 glucose (Fig 4). Similar results were obtained in cells that over-express RecA owing to a mutation in the recA gene SOS operator (Fig 4).…”

“…Cells were grown in either M9 minimal medium supplemented with 0.4% glucose, 5 μM CaCl 2 and 1 mM MgSO 4 or LB medium supplemented with 0.5% glucose at 37°C as described in Cockram et al, 2015 [51]. …”

“…Input and ChIP samples were processed as described in [51]. Adaptor-ligated DNA was size-selected with an average size of ~350 bp Samples that were grown in LB medium were sequenced using 50 bp single-end sequencing on the Illumina HiSeq 2500 platform and samples that were grown in M9 medium were sequenced using 75 bp paired-end sequencing on the Illumina HiSeq 4000 platform.…”

“…All real-time quantitative PCR reactions were performed as described in [51]. All qPCR primers are listed in S3 Table.…”

Precarious maintenance of simple DNA repeats in eukaryotes

“…What about the fact that BIR can lead to significant rearrangements when it occurs between microhomologies on two different chromosomes? The E. coli version of BIR, recombination-dependent replication (RDR), utilizes specifically oriented Chi sites as replication restart sites, ensuring that the strand invasion step starts at non-repetitive 8 bp-long sequences to minimize likelihood of large deletion and insertion events [67,70]. Furthermore, BIR is not cell-cycle sequestered in eukaryotes, and events in G2 can lead to large-scale loss of heterozygosity [71].…”