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

Jakub Wiktor, Marit Van Der Does, Lisa Büller, David J. Sherratt, Cees Dekker

Research output: Contribution to journalArticleScientificpeer-review

15 Citations (Scopus)
19 Downloads (Pure)

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. Realtime imaging shows that RecBCD during end resection degrades DNA with remarkably high speed (i"1.6 kb/s) and high processivity (>7sim;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.

Original languageEnglish
Pages (from-to)1821-1833
Number of pages13
JournalNucleic Acids Research
Volume46
Issue number4
DOIs
Publication statusPublished - 2018

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