Replication fork reversal is rapidly emerging as a pivotal mechanism to explain how stalled or damaged replication forks are processed upon treatment with cancer chemotherapeutics. The first evidence that drug-induced DNA damage induces fork reversal arose from studies using DNA topoisomerase I chemotherapeutic inhibitors. Fork reversal invokes formation of four-way junction structures, reminiscent of Holliday junctions, to prevent the collision of the replication fork with the damage ahead of the fork. These four-way junction structures must be properly resolved once the lesion has been repaired in order to re-establish a functional replication fork and maintain genome stability.
We provided the first mechanistic insight into how replication forks regress and restart as a pivotal response to treatment with DNA topoisomerase I inhibitors. We demonstrated that the human RECQ1 helicase promotes the restart of replication forks that have reversed upon DNA topoisomerase I inhibition, and that the poly(ADPribosyl)ation activity of PARP1 stabilizes forks in their regressed state by limiting their restart by human RECQ1. Having identified a specific and controlled biochemical activity that mediates this process offers new molecular perspectives to potentiate chemotherapeutic regimens based on DNA topoisomerase I inhibitor treatment.
Our current focus is to understand whether replication fork regression and restart is a more general phenomenon that also takes place in the presence of other cancer chemotherapeutics that stall and/or damage replication forks. We are also studying additional factors and alternative mechanisms that might mediate fork reversal and restart depending on the type of agent used to induce replication stress. We combine genome-wide single-molecule replication assays with electron microscopy (EM) approaches to achieve a comprehensive view of the mechanisms of fork reversal and restart. The combination of these approaches allows us to test how selected genotoxic agents affect the dynamics of DNA replication by DNA single-molecule replication assays and simultaneously assess their effect on the frequency of drug-induced reversed forks by EM.
We have also developed efficient assays to study fork reversal and restart in vitro using synthetic oligonucleotides that mimic model replication fork and reversed fork structures. Finally, we have implemented an innovative chromatin immunoprecipitation approach called iPOND (isolation of proteins on nascent DNA) to study the loading of cellular factors at stalled/reversed replication forks. Using this approach, we study how the loading dynamics of the different factors involved in replication fork reversal and restart is affected by the particular agent used to induce replication stress.