Analysis of homologous recombination restarted replication forks in S. pombe

Natural impediments to DNA replication such as replication fork barrier’s (RFB’s), replication-transcription collisions, or secondary structure forming sequences, can result in replication stress and contribute to genomic instability. This project utilizes the Schizosaccharomyces pombe replication fork barrier, RTS1, known to result in replication fork stalling that requires homologous recombination (HR) for restart. The HR- restarted replication fork uses Polymerase ? to synthesise both DNA strands and is known to be error-prone in nature, resulting in both replication fork slippage and gross chromosomal rearrangements. This project produces an optimized system for studying the HR-restarted replication fork at RTS1. By utilizing methods including Pu-Seq, and a genetic replication slippage assay, we are able to investigate both polymerase usage between strands and replication fidelity, respectively. Break induced replication (BIR) in Saccharomyces cerevisiae is another replication fork restart method often compared to HR-restart at RTS1 in S. pombe. Pol32 and its interaction with PCNA are essential for restart using BIR, however, we provide evidence using Cdc27 (ScPol32) that this interaction is important to a lesser extent during HR-restart at RTS1. Additionally, the necessity of the protein Rtf2 for efficient replication fork stalling at RTS1 has been confirmed. Rtf2 and its removal from stalled replication forks has recently been implicated in maintaining genome stability in human cells. Additional methods used including ChIP-qPCR, Co-IP and mass spectrometry has allowed further investigation into proteins involved in the restarted replication fork.