| dc.description.abstract | Efficient and accurate repair of DNA double strand breaks (DSBs) is required to maintain
genomic stability in both eukaryotes and prokaryotes. In Escherichia coli, DSBs are repaired
by homologous recombination (HR). During this process, DNA synthesis needs to be
primed and templated from an intact homologous sequence to restore any information that
may have been lost on the broken DNA molecule. Two critical late stages of the pathway
are repair DNA synthesis and the processing of Holliday junctions (HJs). However, our
knowledge of the detailed mechanisms of these steps is still limited. Our laboratory has
developed a system that permits the induction of a site-specific DSB in the bacterial
chromosome. This break forms in a replication dependent manner on one of the sister
chromosomes, leaving the second sister chromosome intact for repair by HR. Unlike
previously available systems, the repairable nature of these breaks has made it possible to
physically investigate the different stages of DNA double-strand break repair (DSBR) in a
chromosomal context. In this thesis, I have addressed some fundamental questions relating
to repair DNA synthesis and processing of HJs by using a combination of mutants defective
in specific biochemical reactions and an assay that I have developed to detect repair DNA
synthesis, using a polar termination sequence (terB). First, by using terB sites located at
different locations around the break point, it was shown that the DnaB-dependent repair
forks are established in a coordinated manner, meaning that the collision of the repair forks
occurs between two repair DNA synthesis initiation sites. Second, DSBR was shown to
require the PriB protein known to transduce the DNA synthesis initiation signal from PriA
protein to DnaT. Conversely, the PriC protein (known as an alternative to PriB in some
reactions) was not required in this process. PriB was also shown to be required to establish
DnaB-dependent repair synthesis using the terB assay. Third, the establishment and
termination of repair DNA synthesis by collision of converging repair forks were shown to
occur independently of HJ resolution. This conclusion results from the comparison of the
viability of single and double mutants, deficient in either the establishment of DNA
synthesis, HJ resolution or in both reactions, subjected to DSBs and from the study of the
DNA intermediates that accumulated in these mutants as detected by two-dimensional gel
electrophoresis. Fourth, the role of RecG protein during DSB repair was investigated. Solexa
sequencing analyses showed that recG null mutant cells undergoing DSBs accumulate more
DNA around the break point (Mawer and Leach, unpublished data). This phenomenon was
further investigated by two different approaches. Using terB sites in different locations
around the break point and ChIP-Seq analyses to investigate the distribution of RecA in a
recG null mutant demonstrating that the establishment of repair forks depends on the
presence of RecG. Further studies using PriA helicase-dead mutant showed that the interplay
between RecG and PriA proteins is essential for the establishment of correctly oriented
repair forks during DSBR. As a whole, this work provides evidence on the coordinated
nature of the establishment and termination of DNA synthesis during DSBR and how this
requires a correct interplay between PriA-PriB and RecG. A new adapted model of
homologous recombination is presented. | en |
