Replication Protein A Is Required for Meiotic Recombination in Saccharomyces cerevisiae

Abstract

In Saccharomyces cerevisiae, meiotic recombination is initiated by transient DNA double-stranded breaks (DSBs). These DSBs undergo a 5′ → 3′ resection to produce 3′ single-stranded DNA ends that serve to channel DSBs into the RAD52 recombinational repair pathway. In vitro studies strongly suggest that several proteins of this pathway—Rad51, Rad52, Rad54, Rad55, Rad57, and replication protein A (RPA)—play a role in the strand exchange reaction. Here, we report a study of the meiotic phenotypes conferred by two missense mutations affecting the largest subunit of RPA, which are localized in the protein interaction domain (rfa1-t11) and in the DNA-binding domain (rfa1-t48). We find that both mutant diploids exhibit reduced sporulation efficiency, very poor spore viability, and a 10- to 100-fold decrease in meiotic recombination. Physical analyses indicate that both mutants form normal levels of meiosis-specific DSBs and that the broken ends are processed into 3′-OH single-stranded tails, indicating that the RPA complex present in these rfa1 mutants is functional in the initial steps of meiotic recombination. However, the 5′ ends of the broken fragments undergo extensive resection, similar to what is observed in rad51, rad52, rad55, and rad57 mutants, indicating that these RPA mutants are defective in the repair of the Spo11-dependent DSBs that initiate homologous recombination during meiosis.

Meiotic Chromosome Dynamics in Zebrafish

“…It is not known, however, if zebrafish Rad51 and Dmc1 localize to the same or different sites and/or play distinct roles in DSB repair, as reported in several species (Shinohara et al, 1997;Pittman et al, 1998;Yoshida et al, 1998;Howard-Till et al, 2011;Steinfeld et al, 2019;Slotman et al, 2020). In mice, RPA (Replication Protein A complex) is required for DMC/RAD51 recruitment and strand invasion as well as efficient meiotic crossover formation (Sugiyama et al, 1997;Soustelle et al, 2002;Shi et al, 2019;Hinch et al, 2020). Zebrafish RPA likely plays similar roles in meiotic recombination, since it is observed as punctate foci or short stretches mostly at chromosome ends in the leptotene and zygotene stages, and the majority of these signals disappear at the pachytene stage (Moens, 2006;Takemoto et al, 2020).…”

Pathogenic Variations of Homologous Recombination Gene HSF2BP Identified in Sporadic Patients With Premature Ovarian Insufficiency

“…In the prophase of the first meiotic division, DNA double-strand breaks (DSBs) are generated by SPO11 (Keeney et al, 1997). After the modification by MRN, EXO1, and RPA (Soustelle et al, 2002;Lisby et al, 2004;Zakharyevich et al, 2010), the 3′tailed single-strand DNA (ssDNA) of DSBs are recognized by BRCA2, RAD51, and DMC1, which execute the critical step of strand invasion, followed with homologous recombination (HR) and crossover formation (Barlow et al, 1997;Moens et al, 2002;Sharan et al, 2004). HSF2BP is a recently identified meiosis-specific protein, which interacted with BRCA2 and BRME1 to form a complex to mediate the recruitment of RAD51 and DMC1 to the meiotic recombination sites and facilitate the strand invasion (Zhang et al, 2020).…”

Characterization of Replication Protein A homologues from Zea mays

“…These mutants exhibit an abnormal distribution of DSBs and increased 5' DNA resection similar to R~4D52 epistasis group mutants (25). They also exhibit decreased sporulation efficiency and are defective in Spo 11 recombination.…”

“…Since deletions of RPA have been shown to be lethal, these mutants had to be propagated as diploids (25). Strain 4020409 was transformed with complementation plasmids for the expression of the maize RPA 1 homologues, the 4031132 strain was transformed with the ZmRPA2H complementation plasmid following the method described by Schiestle et al (42).…”

“…Mutations in the RFA1 subunit show marked changes in the phenotype of S. cerevisiae. Among these are decreased sporulation efficiency, and viability (25). Yeast genetic complementation has been used to isolate mammalian counterparts for lethal yeast mutant strains (34).…”