An Integrated Molecular, Cellular and Genomic Investigation of DNA Double-Strand Break Repair in Bacteria

    Our laboratory aims to dissect the molecular mechanism of DNA double-strand break repair (DSBR) as it occurs in living E. coli cells.  Despite the decades of research on bacterial DSBR, the application of new technologies to sophisticated in vivo systems for generating DNA breaks is generating new and unexpected insight into this fundamental macromolecular reaction.

    DNA repair pathways are remarkably well conserved from bacteria, through yeasts to humans (see Table below for a list of equivalent recombination proteins).

Table: Recombination proteins in E. coli, S. cerevisiae and Human
                                                                                           

    Creating a repairable DNA double strand break in E. coli

    The primary role of genetic recombination in DSBR is the repair of post-replicative breaks using the identical genetic information present on an undamaged sister chromosome.  However, this has been problematic to investigate at the molecular level in any system because the two recombining partners are genetically identical.  This means that it is difficult to target a DNA double-strand break (DSB) to one sister chromosome at a specific location without risking cleavage of the other sister chromosome at the same site, thereby removing the intact template for repair.  

    We have overcome this problem by developing a system in E. coli where the cleavage by SbcCD nuclease of a DNA hairpin formed on the lagging-strand template at the site of a chromosomal palindrome (lacZ::pal) initiates DSBR following DNA replication, as shown here.  Remarkably, the DSBR event is so efficient that doubling time of these cells is close to unaffected and they only suffer a 0.06% loss of fitness per generation, despite inducing the SOS checkpoint response and increasing in size.

    Visualisation of E. coli cells subjected or not to DSB

    Molecular investigation of DSBR in E. coli

  We have developed sophisticated assays for investigating DSBR in a chromosomal context.  We have used a combination of pulsed field and 2-D native-native gel electrophoresis to analyse the DNA of mutant strains blocked at different stages in the recombination reaction.  Holliday junctions (HJs) formed at a defined locus in the E. coli chromosome have been visualised and shown to accumulate in a ruvAB mutant (see below where arrow heads point out the X-spike of HJs an the Y-arc of replication forks).  Surprisingly, given the previous interpretation that RuvABC and RecG may substitute for each other in the resolution of HJs, these structures did not accumulate in ruv recG double mutants.  Instead, broken DNA was degraded enabling us to conclude that the branch migration activities of RuvAB and RecG provide alternative pathways for stabilising joint molecules and that the nuclease activity of RuvABC is responsible for HJ resolution in E. coli.  This illustrates the power of our system for investigating the structures of recombination intermediates and for clarifying the roles of specific proteins in DSBR.

   Cellular and genomic investigation of DSBR in E. coli

    Our system of live cell imaging is enabling us to visualise the two sides of the DSB (as LacI-Cerulean and TetR-YPet foci) and their association with key proteins as they undergo DSBR.  In addition, we have developed genomic technologies (ChIP-seq and genomic sequencing) to investigate of chromosomal DSBR.