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Beyond double-strand break repair: specific mechanisms of homologous recombination at stressed replication forks.

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

About 10 years ago, breakthrough studies established oncogene-induced replication stress as an early, driving event in tumourigenesis. Deregulated DNA replication fuels genomic instability, driving precancerous lesions into full cellular transformation by inactivation of key DNA damage checkpoint genes. These findings have significantly shifted the research interests in the genome stability field, thus far mainly DSB repair-oriented, towards the mechanisms controlling DNA replication stress. At the same time, the high proliferative surge of cancer cells represents the main biological target of current chemotherapeutic strategy. Moreover, specific genetic defects in mechanisms controlling genome integrity maintenance have been recently exploited to identify druggable targets aimed at selectively killing cancer cells carrying well-defined genetic inactivation. Homologous Recombination (HR) factors, such as BRCA1 and BRCA2, represent a paradigmatic example of tumour suppressor genes, linking genome instability and cancer susceptibility; somatic and acquired mutations in this genes are responsible for approximately 30% of breast and ovarian tumors and the cumulative risk for developing cancer ranged from 49% to 57% in women with BRCA1 or BRCA2 mutations by the age of 70 years. At the same time, genetic defects in these genes confer exquisite sensitivity to clinically relevant chemotherapeutic agents, such as Cisplatin and PARP inhibitors. Nevertheless, the clinical efficacy of these treatments has been limited by the high occurrence of chemoresistence cases.
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.
Among the cellular mechanisms controlling replicative stress, replication fork reversal and restart (fork remodelling) is a general and common transaction aimed at structurally stabilizing the halted fork and promoting template repair/bypass and efficient replication restart. We have previously shown that the central HR factor RAD51 is strictly required to promote fork remodelling. At the same time, stable RAD51 filament formation at reversed forks has been implicated in replication fork protection from unscheduled nuclease-mediated degradation, a clinical-relevant phenomenon recently linked with sensitivity and acquired resistance of HR-deficient tumors to common chemotherapeutics. This finding opens a fascinating scenario, where RAD51 and functionally related Homologous Recombination (HR) factors represent crucial players in maintaining genome stability by remodelling and protecting replication intermediates upon replicative stress, in addition to their established role in mitotic and meiotic Double-Strand Break (DSB) repair.
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.
The results of this project provide the scientific community with a comprehensive genetic map of replication-associated factors involved in the HR-mediated replication stress response, which will represent the groundwork for new lines of investigation. In addition, this research project has unravelled the mechanistic involvement of newly identified HR-related factors in maintaining genome stability upon replicative stress by showing their prominent function in replication fork remodeling and protection, besides their well-established role during DSB repair. Thus, these observations significantly extend the connection between HR deficiency, tumorigenesis and chemotherapeutic sensitivity, a clinical-relevant hot topic for cancer biology, which is currently under deep investigation by multiple research groups. Importantly, a clear molecular understanding of the HR engagement in replication stress would necessarily foster the design of more specific chemotherapeutic strategies towards HR-deficient tumours showing acquired resistance towards Cisplatin or PARP inhibitor. Moreover, it has been recently showed that BRCA2-deficient tumours, which have developed chemoresistence, restore the capacity to protect replication fork, but not their ability to carry out HR-mediated DSB repair, thus making fork degradation a trackable event of high clinical significance. The microscopy-based read-out to follow fork degradation in living cells, which I have started implementing during this project, would certainly represent a powerful tool for the basic and applied research advance in cancer biology.
Poster summarising the RecInRep project