This research aims to establish well-defined model systems to study the mechanisms of DNA replication fork-collapse and fork-restart in eukaryotes. Fork-stalling and fork-collapse are increasingly considered as major sources of genome instability. Because genome instability is often manifested as devastating human diseases such as cancer, it is important to understand how cells deal with such replicative problems. The tools currently available to study replication fork-collapse and subsequent fork-restart in eukaryotes are limited. This proposal aims to study fork-collapse by recreating the precise scenarios that are hypothesized to lead to fork-collapse. The strategy is to exploit the differences in the properties of three different sequence-specific nicking enzymes-AniIK227M, FlpH305L and RepDR189A-to create a single sequence-specific nick in the entire Saccharomyces cerevisiae genome that will be converted into a DSB by an advancing fork. The modified site- specific endonuclease, AnilK227M, creates a simple and easily ligatable nick, which will be used to mimic the scenario wherein a fork fortuitously runs into a nick left behind by incomplete ligation after replication or by DNA repair processes, potentially resulting in fork collapse and genome instability. Nicks created by FlpH305L have a free 5'end but protein-attached 3'end and resemble the lesion created by the antitumor drug camptothecin's inhibition of Topoisomerase I. RepDR189A creates nicks with a free 3'end but protein attached 5'end that resemble inhibition of the Topoisomerase II cleavage complex by the antitumor drug etoposide. The role of putative candidates such as Tdp1 and Sae2 in the removal of protein-DNA complexes will also be ascertained. Two-dimensional gel electrophoresis will be used to visualize replication fork progression across unrepaired nicks. Given that these site-specific nicking enzymes discriminate between Watson and Crick strands, they will be used as tools to investigate lagging strand versus leading strand lesion bypass. It is most likely that leading strand and lagging strand replication deal with template lesions differently. Components of homologous recombination (HR) and post-replication repair (PRR) are known to be critical for replication across irreparable lesions. By means of pedigree analysis, the viability of cells in th event of fork collapse will be tested in HR and PRR mutants to ascertain their roles in dealing with template lesions. Replication associated HR can lead to undesirable outcomes such as translocations, deletions, loss of heterozygosity and copy number variation (CNV) which are clearly linked to genome instability and diseases such as cancer. The potential for fork-collapse to trigger HR will be investigated by an unequal sister chromatid exchange (uSCE) assay. This project will, for the first time, enable a detailed real-time study of the molecular events when a DNA replication fork encounters different types of template strand interruptions.
The goal of this proposal is to study how cells replicate their DNA in the face of site-specific DNA nicks and replication-generated double-strand breaks. Being able to monitor specific lesions by genetic and molecular techniques will help us better understand the mechanisms behind complex gene rearrangements that are characteristic of many human diseases including cancer.
