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Characterization of Physical and Functional Interactions Between the Chromatin Assembly Factor 1, CAF-1, and Homologous Recombination Factors During DNA Repair

Abstract : DNA is constantly exposed to both endogenous and exogenous genotoxic insults. Multiple DNA repair mechanisms are exploited to guard the genome and epigenome stability. Homologous recombination (HR) plays a major role in repairing DNA double strand breaks (DSBs) and restarting stalled replication forks under replicative stress. These two processes are both coupled to chromatin assembly. Chromatin assembly factor 1 (CAF-1) is a highly conserved histone chaperone known to function in a network of nucleosome assembly coupled to DNA repair and replication, by depositing newly synthesized histone (H3-H4)2 tetramers onto the DNA. The fission yeast CAF-1 complex consists of three subunits Pcf1, Pcf2 and Pcf3. CAF-1 has been previously reported to act at the DNA synthesis step during the process of recombination-dependent replication (RDR) and protects the D-loop from disassembly by the RecQ helicase family member, Rqh1. In this study, we addressed the role of CAF-1 during homologous-recombination-mediated DNA repair in fission yeast.Using in vivo and in vitro approaches, we validated interactions within a complex containing Rqh1, CAF-1, PCNA, and Histone H3. We showed that Rqh1 interacts with both Pcf1 and Pcf2 independently of each other, and the Pcf1-Rqh1 interaction is stimulated by DNA damage. We developed an in vivo chromatin binding assay to monitor the association of CAF-1 to the chromatin upon DNA damage. We observed that replication stress but not double strand break favors CAF-1 association to the chromatin. We identified that several HR factors are required for CAF-1 association to the chromatin upon replication stress. In support of this, we have identified physical interactions between Pcf1 and HR factors, including RPA and Rad51. Our data suggest that CAF-1 would associate with the site of recombination-dependent DNA synthesis through physical interactions with HR factors. Put together, this work contributes to strengthening the role of CAF-1 coupled to DNA repair, and reveals the crosstalk between HR factors and chromatin assembly.
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Dingli Dai. Characterization of Physical and Functional Interactions Between the Chromatin Assembly Factor 1, CAF-1, and Homologous Recombination Factors During DNA Repair. Genetics. Université Paris-Saclay, 2018. English. ⟨NNT : 2018SACLS498⟩. ⟨tel-01990602⟩

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