Direct observation of end resection by RecBCD during double-stranded DNA break repair in vivo

Abstract

The formation of 3′ single-stranded DNA overhangs is a first and essential step during homology-directed repair of double-stranded breaks (DSB) of DNA, a task that in Escherichia coli is performed by RecBCD. While this protein complex has been well characterized through in vitro single-molecule studies, it has remained elusive how end resection proceeds in the crowded and complex environment in live cells. Here, we develop a two-color fluorescent reporter to directly observe the resection of individual inducible DSB sites within live E. coli cells. Real-time imaging shows that RecBCD during end resection degrades DNA with remarkably high speed (∼1.6 kb/s) and high processivity (>∼100 kb). The results show a pronounced asymmetry in the processing of the two DNA ends of a DSB, where much longer stretches of DNA are degraded in the direction of terminus. The microscopy observations are confirmed using quantitative polymerase chain reaction measurements of the DNA degradation. Deletion of the recD gene drastically decreased the length of resection, allowing for recombination with short ectopic plasmid homologies and significantly increasing the efficiency of horizontal gene transfer between strains. We thus visualized and quantified DNA end resection by the RecBCD complex in live cells, recorded DNA-degradation linked to end resection and uncovered a general relationship between the length of end resection and the choice of the homologous recombination template.

Live cell imaging reveals that RecA finds homologous DNA by reduced dimensionality search

“…Straight-forward expression of nucleases such as I-SceI or Cas9 can be used to study DNA repair mechanisms (Roukos et al 2013;Lesterlin et al 2014;Anand et al 2017). However, when those nucleases are expressed at low levels in bacteria, cell-to-cell variation in the nuclease concentration leads to cutting of all or no restriction sites, resulting in no repair templates or no breaks (Cui and Bikard 2016;Wiktor et al 2017). To increase control of the DSB induction, we prevent access to the introduced chromosomal I-SceI restriction site (referred from now on as CS) by surrounding it with two lactose operators (lacO), one partly overlapping the restriction site ( Fig 1A-B).…”

“…ParS-Mt1 is bound by several mCherry-ParB and forms a fluorescent focus (Nielsen et al 2006). After a DSB is formed, the RecBCD degrades the parS site and ejects the mCherry-ParB molecules causing a rapid loss of the focus (Wiktor et al 2017).…”

“…The array can be visualized by binding of constitutively expressed MalI-mVenus. This MalI-mVenus focus remains intact during repair as RecBCD resection rarely proceeds that far (Wiktor et al 2017) and thus makes it possible to study the DNA motion after a DSB.…”

“…In a previous E. coli study , chromosomal labels adjacent to a DSB site and its sister were reported to pair about one hour after DSB induction by the I-SceI nuclease (Lesterlin et al 2014). However, it has been demonstrated that such labels are ejected already during end resection (Wiktor et al 2017) which raises questions about the interpretation of the aforementioned pairing. Our results convincingly show that the pairing between distant, segregated sisters in E. coli is rapid and efficient, and involves a loss of the proximal DNA label.…”

“…One of the difficulties in studying the spatial DNA dynamics of the search process in live E. coli cells using fluorescent labels is the potent nuclease activity of RecBCD complex which can degrade many kilobases of DNA before switching activity at a chi site. This makes it impossible to put a DNA binding fluorescent markers in close proximity to the DSB site, as they would be ejected during the initial degradation (Wiktor et al 2017). Moreover, fluorescent protein fusions to RecA tend to decrease or abolish its activity (Renzette et al 2005).…”