A wide variety of inter-dependent DNA repair and cellular signalling pathways, the “DNA Damage Response (DDR)” co-operate to maintain genome stability. DDR pathways suppress the accumulation of genetic changes in the face of endogenous and exogenous genotoxic stress. Genetic (or epigenetic) changes in patients participate in the etiology of cancer. There is also increasing evidence that DNA damage and genome instability in somatic cells contribute to aging. The majority of cancer cells are thought to be defective in one or more DDR proteins, either through mutation of cognate genes during the early stages of carcinogenesis. through the effects of gross chromosomal rearrangements associated with cancer development, or via epigenetic changes. I have chosen to concentrate on the relationship between DNA replication, the DDR and how they contribute to genome instability because a significant proportion of mutations, chromosomal rearrangements and potentially epigenetic changes are generated by either stochastic or DNA damage-induced replication errors. In addition, many therapeutic drugs are specifically genotoxic to replicating cells, a feature that both imparts therapeutic index and results in unwanted side effects on stem cells.
Using fission yeast as a model eukaryote we explore how intra-S checkpoint activation protects the replication fork from inappropriate DNA transactions and, when these pathways fail, how the cell restartes the broken replication fork.
Not all replication barriers are the same and how a specific RFB results in a collapsed fork, and the subsequent mechanisms by which these are repaired, cannot be assumed to equivalent. For example, some RFBs block the replicative helicase, while others interrupt leading or lagging strand polymerases. Such differences provide distinct challenges both to the initial attempts at replisome and fork stabilisation by the intra-S checkpoint and, if forks collapse, appropriate fork processing and/or replication restart.
Fork collapse is a poorly defined term covering a range of poorly understood structures. However, it is clear that the DNA ends of a collapsed fork become vulnerable to additional processing and can be channelled into an inappropriate repair reaction with non-contiguous DNA sequences. The characterised pathways that process and restart collapsed forks in eukaryotes are based on HR. However, HR-dependent restart comes at a price: HR proteins cannot distinguish between sequence homology at the correct site and homology elsewhere. Thus, particularly in the context of repeated sequences, the advantages of restarting a fork come at the expense of an increase in the potential for genome instability.
We have developed a replication fork arrest system by using replication termination sequence 1 (RTS1), a DNA fragment that arrests replication in a site-specific manner whereby fork arrest can be controlled by inducing the DNA binding protein Rtf1. Using this system, we have previously shown that fork restart occurs by an HR-dependent mechanism at the expense of frequent erroneous replication template exchange which results in high levels of gross chromosomal rearrangement . Our recently published data demonstrate that, in addition to the potential template exchange errors made during the restart process (which cause gross chromosomal rearrangements (GCRs)), the forks that are restarted by HR are themselves highly prone to making errors during the subsequent replication. This provides a novel source of replication errors that may promote a significant proportion of genome instability.
Our work is funded by: