Introduction

A wide variety of inter-dependent DNA repair and cellular signaling 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. An early step in the etiology cancer is the activation of an oncogene. This leads to unregulated cell proliferation that is associated with dysregulated DNA replication which results in "oncogene-induced replication stress". This endogenous replication stress can activate checkpoint pathways and promote senescence. However, oncogene-induced replication stress is mutagenic and inactivation of checkpoint pathways in such cells can result in prolonged proliferation and an opportunity for the cells to acquire the further mutations (due to the errors occurring during the endogenously perturbed replication) that are required for full carcinogenesis .

As a result of oncogene-induced replication stress the majority of cancers are thought cells are thought to acquire defects in one or more DDR pathways, either through mutation of cognate genes during the early stages of carcinogenesis. through the effects of gross chromosomal rearrangements associated with cancer development, via epigenetic changes or indirectly via dysregulation of normal cell cycle controls. My laboratory concentrates on the relationship between DNA replication, the DDR and how these relationships contribute to genome instability. In addition to being of importance for understanding the etiology of cancer, understanding the DDR is important in cancer therapy because many therapeutic drugs are specifically genotoxic to replicating cells.

Using fission yeast as a model eukaryote we explore how cells protect and restart replication forks that have encountered obstacles and how this prevents inappropriate DNA transactions that can lead to mutation and gross chromosomal rearrangements.

Not all barriers to DNA replication are the same and how a specific replication fork barrier (RFB) activated the checkpoint, is stabilized (or not)  and how the cell deals with the consequence of fork inactivation are therefore different for different barriers. The subsequent mechanisms by which these lesions are repaired also 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 channeled 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 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. We have also shown that, in addition to the potential template exchange errors made during the restart process (which cause gross chromosomal rearrangements), 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 current work focuses on understanding the DNA processing events that occur during fork restart and defining the mechanism by which replication proceeds after restart.

Our work is funded by the Wellcome Trust