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Final Report Summary - MULTIPLE SEARCH (Mechanism of homology search during homologous recombination)

Alteration to the genome structure fuels cancer development and adaptation, and is at the basis of de novo and inherited congenital disorders. DNA double strand breaks (DSBs) are toxic lesions that endanger cell survival and threaten the stability of the genome. Homologous recombination (HR) is a cellular mechanism that faithfully seals DSBs by templating the repair off an intact identical DNA molecule (Fig. A). To achieve identification of this identical (homologous) sequence amidst the genome, the Rad51 recombinase coats the DNA flanking the break site and harnesses its sequence information to sample nearby dsDNA. Upon homology encounter, a DNA strand exchange intermediate called a displacement loop (D-loop) is formed (Fig. B), from which restoration of the disrupted sequence can be achieved upon DNA synthesis. How the daunting genome-wide search for homology operates remains an upon question. My research project aimed at characterizing the mechanism of homology search and D-loop metabolism in cells, the proteins involved, and identify putative defects with advert consequences for genomic stability.
An important innovation granting the realization of this project has been the development of a suite of techniques (D-loop Capture, D-loop extension, and Multi-invasion Capture) to physically detect key intermediates of the pathway (Piazza et al. Cell 2017; Piazza et al. Methods Enzymol 2018; Piazza et al. Mol. Cell, in revision). These techniques and the kinetic nature of the experimental systems I developed allowed me to make important new discoveries, and are the foundation of several new collaborative projects between the outgoing laboratory (involving two PhD students and one post-doc) and the host laboratory.
First, the D-loop Capture (DLC) assay enabled the study of the kinetics of D-loop formation and the factors involved in strand exchange metabolism and turnover. I found that the majority of D-loops are disrupted in wild type cells, despite being formed at a perfectly homologous donor site. This turnover is enforced by two independent D-loop disruption pathways: one is supported by the Mph1 (human FANCM) helicase and the Sgs1-Top3-Rmi1 (human BLM-TOPO3alpha-RMI1/RMI2) complex, while the other relies on the Srs2 helicase. Apical to these pathways is Rdh54, a conserved Rad54 paralog of poorly characterized function: Rdh54 provides substrate for the Mph1 and Sgs1-Top3-Rmi1 axis, in a catalytic-independent fashion. These results surprisingly suggest that homology search can only be understood in the context of multiple rounds of D-loop formation and disruption (Piazza et al., in revision). This work constitutes the foundation of a general computational homology search framework we are now developing in collaboration with mathematicians (Arsuaga lab). It also identifies multiple proteins with human homologs involved in this new layer of HR control.
Second, I conducted a series of experiments to address our postulated multiplexed homology search model. This model entails that not only the extremity, but the whole length of the DNA flanking the break is used by Rad51 to sample the genome. Fulfilling a prediction of this model is the observation of a “multi-invasion” (MI) intermediate, in which homology has simultaneously been encountered at different positions along the searching DNA molecule (Fig. C). Thanks to a derivative of the DLC assay, I could provide direct evidence for the existence of MI in cells, and uncover certain aspects of their regulation (Piazza et al. Cell 2017).
Third, I postulated that the MI intermediate might be at risk for genomic stability. I devised a genetic assay in yeast to address this possibility, which lead to the discovery of a novel HR-based genomic instability mechanism that we named MI-induced rearrangement (MIR, Fig. D) (Piazza et al. Cell 2017). The MIR mechanism and its regulations have major implications for the research fields of DNA repair, cancer, and meiosis. Notably, the amplification of the initial damage could be involved in the formation of complex “chromothriptic” chromosomal rearrangements observed in cancer genomes (Piazza & Heyer, Trends Cell Biol., in preparation). Furthermore, MIR explains a mounting number of mitotic and meiotic recombination products not accounted for by canonical HR models, thus leading to their revision (Piazza & Heyer, Bioessays 2018).
Finally, I started applying the techniques and concepts developed during the outgoing phase to tackle major open problems in the field of meiotic recombination upon reintegration in the host laboratory (Institut Pasteur). I am currently developing a high-throughput version of the DLC assay and derivatives to perform kinetics analysis of HR intermediates metabolism and crossover formation during synchronous yeast meiosis over a 150 kb chromosomal region. This approach exploits restriction sites features of a synthetic chromosome built in the host laboratory (Muller .. Piazza .. Koszul, Molecular Systems Biology, in press).
In conclusion, my work resulted in (i) the development of assays to study D-loop metabolism in cells that are of general interest for the HR research community, (ii) the characterization of several D-loop disruption activities and their interactions, (iii) the ongoing development of a computational framework for homology search incorporating nascent D-loop turnover, (iv) the demonstration of the existence of a new type of HR intermediate (MI), and (v) the discovery and characterization of a novel genomic instability mechanism rooted in the homology search process (MIR). These findings have major implications for our basic understanding of homologous recombination and its defects underlying simple and complex genomic rearrangements. It is followed upon by three additional PhD students and post-doctoral researchers in the outgoing laboratory, in collaboration with myself in the host laboratory.

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INSTITUT PASTEUR
France

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Life Sciences
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