Mechanisms of replication-induced genome instability
Antony M. Carr1, Izumi Miyabe, Kenichi Mizuno, Yasukazu Daigaku, Saed Mohebi, Johanne M. Murray, Andrea Keszthelyi.
1Genome Damage and Stability Centre, School of Life Sciences, University of Sussex, Brighton, BN1 9RQ, UK
OIS can result in the replisome dissociating from the fork (collapsed fork), or the fork structure to break (broken fork). In these cases, replication restart can proceed by either homologous recombination (HR) or microhomology-primed re-initiation (FoSTeS/MMBIR). Our previous work has demonstrated that HR-restarted DNA replication is intrinsically error prone. We are currently examining the consequences of replicating a region of DNA with a fork that has been correctly restarted by HR. Using DNA polymerase mutants that incorporate excess ribonucleotides we have shown that, following replication restart by HR-dependent mechanisms within S phase, replication is semi-conservative and both strands are synthesised by polymerase delta. We have developed a protocol (Ribo-Seq) to map, genome-wide, the usage of Polymerase delta and Polymerase epsilon. Our data plot, at very high resolution, the location of the replication origins and identify regions of the genome where there is increased usage of polymerase delta to replicate the duplex DNA. Such sites may represent regions prone to genome instability.