Checkpoint Regulation of Replication Forks at Sites of DNA Damage

Abstract

Statement of methods:We have used single‐molecule DNA combing to study how the checkpoint regulates individual origins and forks in response to DNA damage.imageSummary of resultsThe S‐phase DNA damage checkpoint slows replication and allows cells to replicate damaged DNA, but how slowing relates to damage tolerance remains unclear. The standard explanation — that checkpoint delays cell‐cycle progression until the damage can be repaired — does not seem to apply in the case of DNA replication. Instead, replication proceeds in the presence of the damage, but at a slower rate. Replication slowing can be achieved by inhibition of origin firing, slowing of fork progression or a combination of both. In bulk assays, 1uM 4NQO and 0.03% MMS slow S phase to a similar extent. However the combing data shows that 4NQO slows replication by reducing origin firing rate, while MMS slows S phase by stalling forks and reducing the fork rate. While reduction in origin firing rate is checkpoint dependent, reduction in fork rate has checkpoint dependent and independent components. We are currently investigating the mechanistic difference in response to different damaging agents and how the checkpoint regulates fork components to achieve replication slowingAnother key question is how forks replicate quickly through damaged DNA in the absence of the checkpoint. The MMS‐induced lesions we use are believed to stop replicative polymerases. Nonetheless, in the absence of the checkpoint, replication forks pass them without any apparent delay. This bypass seems to be mainly dependent on recombination and translesion polymerase based synthesis. We are working on how forks bypass polymerase‐stalling lesions and what effect this bypass has on genomic stability.Funding Source:NIHGMS