Biology subjects

Guirouilh-Barbat, J.

Publications and source records attributed to Guirouilh-Barbat, J..

5 recordsLinked to original sources

bioRxiv2023

Polθ is phosphorylated by Polo-like kinase 1 (PLK1) to enable repair of DNA double strand breaks in mitosis

DNA double strand breaks (DSBs) are deleterious lesions that challenge genome integrity. To mitigate this threat, human cells rely on the activity of multiple DNA repair machineries that are tightly regulated throughout the cell cycle1. In interphase, DSBs are mainly repaired by non-homologous end joining (NHEJ) and homologous recombination (HR)2. However, these pathways are completely inhibited in mitosis3-5, leaving the fate of mitotic DSBs unknown. Here we show that DNA polymerase theta (Pol{theta})6 repairs mitotic DSBs and thereby maintains genome integrity. In contrast to other DSB repair factors, Pol{theta} function is activated in mitosis upon phosphorylation by the Polo-like kinase 1 (PLK1). Phosphorylated Pol{theta} is recruited to mitotic DSBs, where it mediates joining of broken DNA ends, while halting mitotic progression. The lack of Pol{theta} leads to a shortening of mitotic duration and defective repair of mitotic DSBs, resulting in a loss of genome integrity. In addition, we identify mitotic Pol{theta} repair as the underlying cause of the synthetic lethality between Pol{theta} and HR. Our findings reveal the critical importance of mitotic DSB repair for maintaining genome stability.

cell biology↗

bioRxiv2022

Human RAD52 stimulates the RAD51-mediated homology search

Homologous recombination (HR) is a DNA repair mechanism of double strand breaks and blocked replication forks that involves a process of homology search and homologous pairing leading to the formation of synaptic intermediates whose architecture and dynamics are tightly regulated to ensure genome integrity. In this mechanism, RAD51 recombinase plays a central role and is supported by many partner including BRCA2 and RAD52. If the mediator function of BRCA2 to load RAD51 on RPA-covered ssDNA is well established, the role of RAD52 in HR, more precisely its interplay with BRCA2 is still far from understood. We have used Transmission Electron Microscopy combined with biochemistry to characterize the sequential participation of RPA, RAD52 and BRCA2 in the assembly of the RAD51 filament, its architecture and its activity. Despite our results confirm that RAD52 lacks a mediator activity, we observed that RAD52 can tightly bind to RPA-coated ssDNA, inhibit the mediator role of BRCA2 and form shorter RAD52- and RAD51-containing mixed filaments that are more efficient in subsequent homology search and formation of synaptic complexes and D-loops, resulting in more frequent multi-invasions as well. Through the characterization of the behavior of RAD52 and BRCA2, these results provide new molecular insights on the formation and regulation of presynaptic and synaptic intermediates during human HR.

biochemistry↗

bioRxiv2022

In vivo inactivation of RAD51-mediated homologous recombination leads to premature aging, but not to tumorigenesis

Genetic instability is a hallmark of both cancer and aging. Homologous recombination (HR) is a prominent DNA repair pathway maintaining genomic integrity. Mutations in many HR genes lead to cancer predisposition. Paradoxically, the consequences of mutations in the pivotal HR player, RAD51, on cancer development remain puzzling. Moreover, in contrast with other HR genes, RAD51 mouse models are not available to experimentally address the role of RAD51 on aging and carcinogenesis, in vivo. Here, we engineered a mouse model with an inducible dominant negative form of RAD51 (SMRad51) that suppresses RAD51-mediated HR without stimulating alternative non-conservative repair pathways. We found that, in vivo expression of SMRad51 did not trigger tumorigenesis, but instead induced premature aging. We propose that these in vivo phenotypes result from the exhaustion of proliferating progenitors submitted to chronic endogenous replication stress resulting from RAD51-mediated HR suppression. Our data underline the importance of the RAD51 activity for progenitors homeostasis, preventing aging, and more generally for the balance between cancer and aging.

cancer biology↗

bioRxiv2019

Mammalian RAD51 prevents non-conservative alternative end-joining and single strand annealing through non-catalytic mechanismssms

The selection of the DNA double-strand breaks (DSBs) repair pathway is decisive for genetic stability/instability. We proposed that it acts according to two successive steps: 1-canonical non-homologous end-joining (C-NHEJ) versus single-strand DNA (ssDNA) resection; 2- on ssDNA, gene conversion (GC) versus non-conservative single-strand annealing (SSA) or alternative end-joining (A-EJ).\n\nUsing intramolecular substrates, we systematically analysed the equilibrium between the different DSB repair pathways. We show that ablation of RAD51 stimulated both SSA and A-EJ but did not stimulate C-NHEJ, validating the two-step model. Moreover, we found that two ATP-mutant dominant-negative forms of RAD51 that stimulated non-conservative repair, failed to load into damaged chromatin, clarifying the role of ATP in RAD51-mediated HR, also. In contrast, another dominant-negative form of RAD51, which retains its DNA binding capacities, repressed SSA and A-EJ, revealing two separable functions of RAD51 i.e. GC and non-conservative repair inhibition. In vitro assays show that the binding of RAD51 on both complementary ssDNA is required to block both spontaneous and RAD52-induced strand annealing. Therefore, RAD51 represses non-conservative repair (SSA and A-EJ), by inhibiting the annealing step through ssDNA occupancy, independently of the catalytic strand-exchange activity required for GC.

molecular biology↗

bioRxiv2019

RAD51 promotes non-homologous genetic rearrangements that are prevented by 53BP1

Homologous recombination (HR), which requires long sequence homologies, is considered a high fidelity mechanism, preserving genome stability. In contrast, we show here that the central HR players RAD51 or BRCA2, promote genetic instability, fostering translocations and capture of ectopic chromosomal sequences when joining distant DNA breaks. Surprisingly, these events do not involve sequence homologies. Moreover, our data reveal that 53BP1 protects against RAD51-mediated non-homologous genetic rearrangements. Finally, analysis of a large panel of breast tumors revealed that BRCA2 proficiency is associated with increased frequency of capture of non-homologous sequences at junctions of structural variants (translocations, duplications, inversions, deletions). These data reveal that HR proteins (RAD51, BRCA2) possess the intrinsic capacity to generate genetic instability through sequence homology-independent processes, and that 53BP1 protects against it. We propose that BRCA2/RAD51-mediated genome instability occurs in the course of sequence homology search for HR.

molecular biology↗