The DNA helicase FANCJ (BRIP1) functions in double strand break repair processing, but not crossover formation during prophase I of meiosis in male mice

Meiotic recombination between homologous chromosomes is initiated by the formation of hundreds of programmed double-strand breaks (DSBs). Approximately 10% of these DSBs result in crossovers (COs), sites of physical DNA exchange between homologs that are critical to correct chromosome segregation. Virtually all COs are formed by coordinated efforts of the MSH4/MSH5 and MLH1/MLH3 heterodimers, the latter representing the defining marks of CO sites. The regulation of CO number and position is poorly understood, but undoubtedly requires the coordinated action of multiple repair pathways. In a previous report, we found gene-trap disruption of the DNA helicase, FANCJ (BRIP1/BACH1), elicited elevated numbers of MLH1 foci and chiasmata. In somatic cells, FANCJ interacts with numerous DNA repair proteins including MLH1, and we hypothesized that FANCJ functions with MLH1 to regulate the major CO pathway. To further elucidate the meiotic function of FANCJ, we produced three new Fancj mutant mouse lines via CRISPR/Cas9 gene editing: a full-gene deletion, truncation of the N-terminal Helicase domain, and a C-terminal dual-tagged allele. We also generated an antibody against the C-terminus of the mouse FANCJ protein. Surprisingly, none of our Fancj mutants show any change in either MLH1 focus counts during pachynema or total CO number at diakinesis of prophase I. We find evidence that FANCJ and MLH1 do not interact in meiosis; further, FANCJ does not co-localize with MSH4, MLH1, or MLH3 in meiosis. Instead, FANCJ co-localizes with BRCA1 and TOPBP1, forming discrete foci along the chromosome cores beginning in early meiotic prophase I and densely localized to unsynapsed chromosome axes in late zygonema and to the XY chromosomes in early pachynema. Fancj mutants also exhibit a subtle persistence of DSBs in pachynema. Collectively, these data indicate a role for FANCJ in early DSB repair, but they rule out a role for FANCJ in MLH1-mediated CO events. Errors during meiotic chromosome segregation can result in aneuploidy in offspring, a leading cause of birth defects, pregnancy loss, and infertility. Meiotic crossovers (COs), exchanges of genetic material between parental chromosomes, are critical to ensuring faithful segregation. Nearly all COs are formed by the mismatch repair proteins, MSH4/MSH5 and MLH1/MLH3. Although the number of COs is highly controlled, the mechanisms underpinning this regulation remain poorly understood. We previously found that disruption of the DNA Helicase, FANCJ, caused an increase in CO number. In somatic cells, FANCJ is known to function in numerous DNA repair pathways and interact with diverse repair proteins, including MLH1. Thus, we hypothesized FANCJ functions with MLH1 to regulate meiotic CO formation. In this study we report that, surprisingly, FANCJ does not interact with MLH1 in mouse meiosis. Instead, FANCJ appears earlier during meiosis and does not spatially coincide with MLH1 or MSH4. Further, loss of FANCJ has no impact on CO number, but does cause an increase of unrepaired DNA breaks in late meiotic prophase. These results indicate that FANCJ is dispensable for CO formation, but likely functions in early DNA repair processes that are nonetheless important for maintaining germ cell genomic integrity.