Abstract
Homologous recombination (HR) is a faithful DNA repair mechanism that repairs DNA double-strand breaks (DSB) in a template-dependent manner. HR plays a critical role in meiosis for ensuring proper chromosome segregation and in promoting genetic diversity. Meiotic recombination initiates with the formation of programmed double-strand breaks (DSB) by SPO11 in early meiotic prophase. The DSB ends are then resected and resulting 3’ single-stranded DNA (ssDNA) tails are protected by RPA. Strand exchange proteins RAD51 (ubiquitous) and DMC1 (meiosis specific) replace RPA on ssDNA. RAD51and/or DMC1 bound on ssDNA forms a nucleoprotein filament that carries out homology search and the strand invasion of an intact duplex DNA. This strand invasion step is central to meiotic recombination and is tightly regulated by the balance between several proteins that promote or oppose the loading and stable assembly of RAD51/DMC1 on ssDNA and the subsequent reaction of strand invasion. The SWS1-SWSAP1 complex (Shu complex) is one of these factors that stabilize the RAD51/DMC1 filament. Recent studies in Arabidopsis and human cells proposed FIGNL1-FLIP as a novel, conserved negative regulator of RAD51 and DMC1.The goal of my PhD project was to characterize the functional role of FIGNL1 and FLIP during meiotic recombination in mice, by generating and characterizing mouse models of male germline-specific, conditional inactivation (conditional knockout) of Fignl1 and Flip genes (Fignl1 cKO, Flip cKO). I showed that these two genes are essential for fertility: their absence causes spermatocytes to stop and be eliminated before meiotic divisions. Fignl1cKO and FlipcKO show similar defects in homologous chromosome synapsis and meiotic DSB repair. In the absence of FIGNL1 or FLIP, DSBs are formed and resected (RPA foci are present on chromosome axes), but their repair by HR does not succeed. In WT mice, RAD51 and DMC1 form transient foci on meiotic chromosomes, visualizing recombination intermediates. In Fignl1 cKO and Flip cKO spermatocytes on the contrary, RAD51 and DMC1 foci accumulate in greater numbers, and they also form fiber-like structures never observed in WT. MSH4, which is a marker of an HR step subsequent to strand exchange reaction catalyzed by DMC1 with RAD51, normally forms foci on meiotic chromosomes after DMC1 and RAD51. The number of MSH4 foci is greatly reduced in both mutants, indicating a lack of progression in the HR process. The DMC1 and RAD51 proteins, although they are recruited to the chromosomes, may therefore not effectively catalyze the strand exchange reaction in the absence of FIGNL1 or FLIP.In the absence of the functional SPO11 protein, meiotic DSBs are not formed and RAD51 and DMC1 are not recruited to the chromosomes. However, in Spo11 Fignl1 cKO and Spo11 Flip cKO double mutants, RAD51 and DMC1 accumulate on the chromosomes as much as in Fignl1 cKO and Flip cKO single mutants. In the absence of FIGNL1 or FLIP, RAD51 and DMC1 are therefore recruited to the meiotic chromosome independently of DSBs. The FIGNL1-FLIP complex might restrict or prevent the stable association of DMC1 and RAD51 with intact double-stranded DNA, and also with structural proteins of the meiotic chromosome.Altogether, my work demonstrates that the conserved FIGNL1-FLIP complex is an essential regulator of meiotic HR that limits the accumulation of RAD51 and DMC1 on meiotic chromosomes. It plays a role, direct or indirect, in the normal process of meiotic recombination, reflected by a defect in progression towards the late stages of HR in Fignl1 cKO and Flip cKO spermatocytes.