Rad51 and Rad54 promote noncrossover recombination between centromere repeats on the same chromatid to prevent isochromosome formation

Abstract

Centromeres consist of DNA repeats in many eukaryotes. Non-allelic homologous recombination (HR) between them can result in gross chromosomal rearrangements (GCRs). In fission yeast, Rad51 suppresses isochromosome formation that occurs between inverted repeats in the centromere. However, how the HR enzyme prevents homology-mediated GCRs remains unclear. Here, we provide evidence that Rad51 with the aid of the Swi/Snf-type motor protein Rad54 promotes non-crossover recombination between centromere repeats to prevent isochromosome formation. Mutations in Rad51 and Rad54 epistatically increased the rates of isochromosome formation and chromosome loss. In sharp contrast, these mutations decreased gene conversion between inverted repeats in the centromere. Remarkably, analysis of recombinant DNAs revealed that rad51 and rad54 increase the proportion of crossovers. In the absence of Rad51, deletion of the structure-specific endonuclease Mus81 decreased both crossovers and isochromosomes, while the cdc27/pol32-D1 mutation, which impairs break-induced replication, did not. We propose that Rad51 and Rad54 promote non-crossover recombination between centromere repeats on the same chromatid, thereby suppressing crossover between non-allelic repeats on sister chromatids that leads to chromosomal rearrangements. Furthermore, we found that Rad51 and Rad54 are required for gene silencing in centromeres, suggesting that HR also plays a role in the structure and function of centromeres.

Conditional knockout of RAD51-related genes inLeishmania majorreveals a critical role for homologous recombination during genome replication

“…These loci might then be vulnerable to breakage, such as during chromosome segregation, and RAD51-directed HR may be required to repair any breaks, such as after mitosis in order to allow proper licensing and firing of origins at these loci. Such a scenario could be compatible with data from other eukaryotes of important roles for RAD51-directed HR is maintaining centromere function (77)(78)(79). In T. brucei, the presence of further ORC-defined origins in each chromosome may compensate for loss of RAD51 causing impaired centromere-focused origin function, but such a mutation in Leishmania might be more detrimental, given the presence of only a single major centromere-focused origin in each chromosome (39).…”

Regulation of mitotic recombination between DNA repeats in centromeres

“…Rad51-dependent HR is important to maintain genome integrity, as mutations in BRCA2 elevate GCR events, resulting in predisposition to breast and ovarian cancer ( 34 ). rad51 mutations reduce gene conversion and instead increase homology-mediated GCRs ( 35 – 37 ). Besides its role in Rad51 filament formation, Rad52 plays a role in Rad51-independent HR.…”

“…In previous studies, using fission yeast we have demonstrated that Rad51 and Rad54 channel spontaneous recombination between centromere inverted repeats into a non-crossover pathway, thereby limiting crossovers that result in centromere inversion or isochromosome formation ( 35 ). Here, we compared mitotic recombination between inverted repeats in centromere and in arm regions.…”

Rdh54/Tid1 Inhibits Rad51-Rad54-Mediated D-loop Formation and Limits D-loop Length

“…Moreover, we conclude that Tid1-KR cannot dismantle D-loops after they are formed. D-loop formation by Rad54 requires its ATPase activity in vivo (Onaka et al, 2016) and in vitro (Tavares et al, 2019). The Rad54 ATPase activity is stimulated by dsDNA-Rad51 .…”