Periodic Reporting for period 1 - RecInRep (Beyond double-strand break repair: specific mechanisms of homologous recombination at stressed replication forks.)
Reporting period: 2016-04-01 to 2018-03-31
Besides the well-established role of HR factors in classical double-strand breaks (DSB) repair, a growing body of literature has recently pointed towards a more penetrant, clinically-relevant role in managing DNA replication stress. By using an integrated, multidisciplinary experimental strategy, combining microscopy-based high-throughput screenings with single-molecule and biochemical approaches, this project has been conceived to mechanistically dissect the role of HR during replication stress, besides its well-characterised function in DSB repair, and to identify new HR factors specifically involved in this process.
This project has significantly helped to define the molecular basis of cancers harbouring HR-defective genetic background, but it also provides the groundwork for the identification of new targets to be exploited to selectively kill cancer cells or to overcome their acquired resistance to common chemotherapeutic strategies.
The main goal of the project has been to mechanistically dissect the roles of RAD51 and other HR factors in fork remodelling and to identify new HR factors specifically involved in the response to replication stress, beyond their role in DSB repair. In the first part of the project I have implemented and carried out a microscopy-based, high-content siRNA genetic screen aimed at identifying replication-associated factors that positively or negatively control RAD51 foci formation upon mild treatment of genotoxic agents, which stall the replicative process without triggering detectable DSB. The multicolor imaging strategy of the genetic screen has been designed to monitor several different parameters, besides the main read-out, such as cell cycle, proliferation and DSB effects associated with each specific genetic manipulation, thus providing a high load of information and allowing us a stringent result analysis. Factors whose depletion decrease or increase RAD51 foci formation by at least two siRNAs were respectively classified by the relative Z-score as positive (Z-score <1.5) or negative (Z-score >1.5) regulators and selected for further analysis. By a combination of secondary, microscopy-based screens using other replication stress-associated read-outs, validation experiments by additional siRNAs and rescue experiments and single-molecule DNA fiber assay, the most interesting factors displaying direct involvement in HR-mediated replicative stress response were selected and characterized with more specialized techniques during the second part of the project. In particular, Electron Microscopy (EM) visualization of replication intermediates extracted from actively proliferating cells has been used to monitor fork remodelling and protection events and draw a mechanistic model for the molecular role of the newly identified factors in RAD51- and BRCA2-mediated replication fork and genome stability maintenance.