Abstract
Accurate repair of DNA double-strand breaks (DSBs) is crucial for cell survival and genome integrity. In Escherichia coli, DSBs are repaired by homologous recombination (HR), using an undamaged sister chromosome as template. The DNA intermediates of this pathway are expected to be branched molecules that may include 4-way structures termed Holliday junctions (HJs), and 3-way structures such as D-loops and repair forks. Using a tool creating a site-specific, repairable DSB on only one of a pair of replicating sister chromosomes, we have determined how these branched DNA intermediates are distributed across a DNA region that is undergoing DSB repair. In cells, where branch migration and cleavage of HJs are limited by inactivation of the RuvABC complex, HJs and repair forks are principally accumulated within a distance of 12 kb from sites of recombination initiation, known as Chi, on each side of the engineered DSB. These branched DNA structures can even be detected in the region of DNA between the Chi sites flanking the DSB, a DNA segment not expected to be engaged in recombination initiation, and potentially degraded by RecBCD nuclease action. This is observed even in the absence of the branch migration and helicase activities of RuvAB, RadA, RecG, RecQ and PriA. The detection of full-length DNA fragments containing HJs in this central region implies that DSB repair can restore the two intact chromosomes, into which HJs can relocate prior to their resolution. The distribution of recombination intermediates across the 12kb region beyond Chi is altered in xonA, recJ and recQ mutants suggesting that, in the RecBCD pathway of DSB repair, exonuclease I stimulates the formation of repair forks and that RecJQ promotes strand-invasion at a distance from the recombination initiation sites.
Highlights
The repair of DNA double-strand breaks (DSBs) is crucial for cell viability and its accuracy is important to prevent genome rearrangements
In the bacterium Escherichia coli, these breaks are repaired by the RecBCD pathway of homologous recombination, which involves copying genetic information from another intact and identical DNA template
The broken DNA and the template DNA form 4-way joint DNA molecules called Holliday junctions, and information lost from the broken chromosome is restored by DNA replication at three-way repair forks
Summary
The repair of DNA double-strand breaks (DSBs) is crucial for cell viability and its accuracy is important to prevent genome rearrangements. RecBCD loads the DNA binding protein RecA onto these overhangs [9] and RecA forms nucleoprotein filaments, which, during the synapsis phase, initiate homology search and invade homologous duplex DNA to form 3-way structures called D-loops [5]. These D-loops mature into 4-way DNA structures termed Holliday junctions (HJs) [10] and the two chromosomes joined by these branched molecules, need to be separated. The structure and the distribution of the late recombination intermediates of the pathway can shed light on the roles of proteins that are key to this pathway
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