Structure and distribution of homologous recombination intermediates formed during Escherichia coli DNA double-strand break repair

Accurate repair of DNA double-strand breaks (DSBs) is crucial for both cell survival and genome integrity. In Escherichia coli, these breaks are repaired by the homologous recombination (HR) pathway, normally using an undamaged sister chromosome as template. The DNA intermediates of this pathway are expected to be branched molecules that may include Holliday junctions (HJs), D-loops and replication forks. Due to the transient nature of these intermediates and the overlapping action of the pathway proteins, it is difficult to determine the mode and extent of action of the proteins in vivo. Towards that purpose, the study of the structure and distribution of branched repair intermediates that can be detected in recombination mutants is vital. For this, I have optimised the stabilisation and isolation of HR intermediates, using a specific DSB system (palindrome/SbcCD). This system allows the introduction of a site-specific, repairable DSB on only one of a pair of replicating sister chromosomes, leaving the second sister chromosome intact for repair by HR. This study has shown that in cells where branch migration and cleavage of HJs are limited by inactivation of the RuvABC complex, HJs and replication forks are principally accumulated within a distance of 12 kb from sites of recombination initiation, known as Chi, located on both sides of the engineered DSB. Limited movement of these HJs is observed in the presence of branch migration by RuvAB but in absence of RuvC. Surprisingly, these branched DNA structures can even be detected in the central 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 branch migration by RuvAB and RadA and of the helicase activities of RecG, RecQ and PriA. The detection of DNA fragments, of expected electrophoretic mobility, 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. HJs and replication forks in this central region display a constrained distribution, suggesting that these structures are confined in some way by the DSB site itself. Exonuclease I seems to play a role in converting D-loops into repair forks and RecJ seems to promote strand invasion further away from the recombination initiation sites. Using a strategy based on a synthetic region of chromosomal DNA devoid of sites for 4-base cutting restriction enzymes, I have also optimised a protocol for purifying DSB repair intermediates from a single chromosomal location for analysis by transmission electron microscopy (TEM). This promises to enable the direct visualisation of recombination intermediates and help infer the mechanism of their formation. All in all, my investigation into the structure and distribution of DNA repair intermediates, formed across a DSB region, in the absence of the HJ branch migration and resolution complex RuvABC, sheds light on how far initial events like resection and strand invasion following a site-specific DSB can extend in vivo, and how these initial events can lead to the accumulation of branched structures in a central region of DNA not engaged in these initial events. The impact, or lack of impact, of additional mutations in genes implicated in recombination (e.g. recG, recQ, radA, priA, recJ, xonA) place constraints on models of DSB repair by homologous recombination in E. coli