4 research outputs found
RecBCD coordinates repair of two ends at a DNA double-strand break, preventing aberrant chromosome amplification
DNA double-strand break (DSB) repair is critical for
cell survival. A diverse range of organisms from bacteria to humans rely on homologous recombination
for accurate DSB repair. This requires both coordinate action of the two ends of a DSB and stringent
control of the resultant DNA replication to prevent
unwarranted DNA amplification and aneuploidy. In
Escherichia coli, RecBCD enzyme is responsible for
the initial steps of homologous recombination. Previous work has revealed recD mutants to be nuclease
defective but recombination proficient. Despite this
proficiency, we show here that a recD null mutant is
defective for the repair of a two-ended DSB and that
this defect is associated with unregulated chromosome amplification and defective chromosome segregation. Our results demonstrate that RecBCD plays
an important role in avoiding this amplification by
coordinating the two recombining ends in a manner
that prevents divergent replication forks progressing
away from the DSB site
A perfect palindrome in the Escherichia coli chromosome forms DNA hairpins on both leading- and lagging-strands
DNA palindromes are hotspots for DNA double strand breaks, inverted duplications and intra-chromosomal translocations in a wide spectrum of organisms from bacteria to humans. These reactions are mediated by DNA secondary structures such as hairpins and cruciforms. In order to further investigate the pathways of formation and cleavage of these structures, we have compared the processing of a 460 base pair (bp) perfect palindrome in the Escherichia coli chromosome with the same construct interrupted by a 20 bp spacer to form a 480 bp interrupted palindrome. We show here that the perfect palindrome can form hairpin DNA structures on the templates of the leading- and lagging-strands in a replication-dependent reaction. In the presence of the hairpin endonuclease SbcCD, both copies of the replicated chromosome containing the perfect palindrome are cleaved, resulting in the formation of an unrepairable DNA double-strand break and cell death. This contrasts with the interrupted palindrome, which forms a hairpin on the lagging-strand template that is processed to form breaks, which can be repaired by homologous recombination
RecG directs DNA synthesis during double-strand break repair
Homologous recombination provides a mechanism of DNA double-strand break repair (DSBR) that requires an intact, homologous template for DNA synthesis. When DNA synthesis associated with DSBR is convergent, the broken DNA strands are replaced and repair is accurate. However, if divergent DNA synthesis is established, over-replication of flanking DNA may occur with deleterious consequences. The RecG protein of Escherichia coli is a helicase and translocase that can re-model 3-way and 4-way DNA structures such as replication forks and Holliday junctions. However, the primary role of RecG in live cells has remained elusive. Here we show that, in the absence of RecG, attempted DSBR is accompanied by divergent DNA replication at the site of an induced chromosomal DNA double-strand break. Furthermore, DNA double-stand ends are generated in a recG mutant at sites known to block replication forks. These double-strand ends, also trigger DSBR and the divergent DNA replication characteristic of this mutant, which can explain over-replication of the terminus region of the chromosome. The loss of DNA associated with unwinding joint molecules previously observed in the absence of RuvAB and RecG, is suppressed by a helicase deficient PriA mutation (priA300), arguing that the action of RecG ensures that PriA is bound correctly on D-loops to direct DNA replication rather than to unwind joint molecules. This has led us to put forward a revised model of homologous recombination in which the re-modelling of branched intermediates by RecG plays a fundamental role in directing DNA synthesis and thus maintaining genomic stability
DNA synthesis during double-strand break repair in escherichia coli
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