Global analysis of double-strand break processing reveals in vivo properties of the helicase-nuclease complex AddAB

Abstract

In bacteria, double-strand break (DSB) repair via homologous recombination is thought to be initiated through the bi-directional degradation and resection of DNA ends by a helicasenuclease complex such as AddAB. The activity of AddAB has been well-studied in vitro, with translocation speeds between 400-2000 bp/s on linear DNA suggesting that a large section of DNA around a break site is processed for repair. However, the translocation rate and activity of AddAB in vivo is not known, and how AddAB is regulated to prevent excessive DNA degradation around a break site is unclear. To examine the functions and mechanistic regulation of AddAB inside bacterial cells, we developed a next-generation sequencingbased approach to assay DNA processing after a site-specific DSB was introduced on the chromosome of Caulobacter crescentus. Using this assay we determined the in vivo rates of DSB processing by AddAB and found that putative chi sites attenuate processing in a RecA-dependent manner. This RecA-mediated regulation of AddAB prevents the excessive loss of DNA around a break site, limiting the effects of DSB processing on transcription. In sum, our results, taken together with prior studies, support a mechanism for regulating AddAB that couples two key events of DSB repair-the attenuation of DNA-end processing and the initiation of homology search by RecA-thereby helping to ensure that genomic integrity is maintained during DSB repair. Author summaryDouble-strand breaks (DSBs) are a threat to genome integrity and are faithfully repaired via homologous recombination. The initial processing of DSB ends that prepares them for recombination has been well-studied in vitro, but is less well characterized in vivo. We describe a deep sequencing-based assay for assessing the early steps of DSB processing in bacterial cells by the helicase-nuclease complex AddAB. We find that a combination of chi site recognition and RecA loading is required to attenuate AddAB activity. In the PLOS Genetics | https://doi.org/10.1371/journal.pgen

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

“…In order to verify that the large extent and the asymmetry of the end resection were not caused or distorted by the imaging conditions or by the presence of fluorescent ParB proteins, we developed a qPCR-based method to independently monitor the progression of RecBCD in a population of synchronously resecting E. coli cells. A similar qPCR-based methodology was used before to study resection in yeast cells ( 34 ) and recently in Caulobacter cresentus ( 35 ). Using qPCR probes, we measured the concentrations of chromosomal markers in the proximity of the DSB as they were processed by RecBCD (Figure 4A ).…”

“…Speeds of eukaryotic and prokaryotic end resection were estimated before from indirect measurements such as southern blots, DAPI signal decay or quantitative modeling to fit degradation profiles obtained with high-throughput sequencing. Those speed values were reported to be 1 bp/s for vegetative yeast cells ( 42 ), 10 bp/s for yeast mitotic resection ( 43 ), 190 bp/s for E. coli ( 13 ) and 400 bp/s for C. cresentus ( 35 ). Direct measurements of the dynamics of end resection in different species would be of future interest, to explore why some species invest in a very fast end-processing, whereas some others seem to be satisfied with much lower speeds.…”

SMC protein RecN drives RecA filament translocation and remodelling for in vivo homology search

“…To follow RecA-dependent search dynamics in vivo, we use the I-SceI system developed in Caulobacter crescentus (4,12) that allows us to visualize DSB repair between distant homologous regions (Fig. S1A).…”

Modulation of RecFORQ- and RecA-mediated homologous recombination inEscherichia coliby isoforms of translation initiation factor IF2

“…We further suggest that loss of IF2-1 leads to reduced exonuclease V action (which is one component of RecBCD function) after DSBs are generated (3,8,11), resulting in reduced DNA degradation. Should this postulated second effect of IF2-1 deficiency be in some way a consequence of the first, it may point to existence of an interesting phenomenon of retrograde control of RecBCD nuclease function by the RecA nucleoprotein filament, an idea that has previously been suggested for Caulobacter crescentus (whose RecBCD equivalent is AddAB) (96). The proposed second effect could also explain why loss of IF2-1 is associated with a log-scale decrease in efficiency of two-ended DSB repair whereas its effects in the HR assays were more modest.…”