The Replication Factor A2 N‐Terminus Is Required for Proper Progression Through Meiotic Divisions
Authors: Adsero Angela , Wilson Timothy , Haring Stuart 
Published: November 2023

Meiosis is an essential process in sexually‐reproducing organisms for the generation of haploid gametes. In humans, defects in meiosis are known to lead to cellular diseases caused by nondisjunction (e.g., Down Syndrome and Turner Syndrome), translocation, or loss/gain of parts of chromosomes. Since many of the proteins involved in the meiotic process are conserved from humans to yeast, the budding yeast Saccharomyces cerevisiae provides a model system for the induction and study of meiosis. During meiosis, programmed double‐stranded breaks (DSBs) occur at a high frequency throughout the genome, initiating meiotic recombination and ensuring proper chromosome segregation. Due to 5′‐>3′ resection of the breaks, single‐stranded DNA (ssDNA) intermediates are formed. A heterotrimeric protein complex, called Replication Protein A (RPA), binds to the ssDNA to stabilize and direct its processing, thereby protecting the integrity of the genome.

Replication Protein A is conserved in yeast (called Replication Factor A; RFA) and is composed of three subunits: Rfa1 (70 kDa), Rfa2 (32 kDa), and Rfa3 (14 kDa). Replication Factor A is essential for replication, repair/recombination, and cell cycle regulation in mitotically‐growing cells. RFA is also required in meiosis, as an rfa1‐t11 (K45E) mutant displays reduced sporulation, low spore viability, and reduced meiotic recombination. In human cells, the Rpa2 N‐terminus (NT) is hyper‐phosphorylated in response to DNA damage; however, the contribution of this domain in meiosis has yet to be determined.

To elucidate the role of the Rfa2 NT in meiosis, Rfa2 NT mutants were generated that either mimic hyper‐phosphorylation (rfa2‐Dx) or are non‐phosphorylatable (rfa2‐Ax and rfa2‐DNx). Preliminary data shows that the Rfa2 NT appears to play a role in meiosis. Both rfa2‐Dx and rfa2‐Ax mutants consistently sporulate similarly to wild‐type (WT) cells, while rfa2‐DNx cells exhibit drastically reduced sporulation efficiency. Furthermore, the Rfa2 mutant cells' spore viability is only greatly affected when the NT domain is removed. This indicates that the domain is necessary, but that its phosphorylation state may only have a marginal role during meiosis. The defect in meiosis does not appear to be due to defective homologous recombination (HR), although this is currently being confirmed by a second method. Rather, we suggest that the presence of Rfa2 NT may have a role in proper exit from the pachytene checkpoint and/or a direct role in meiotic chromosomal division(s). This is based on the observation that the rfa2‐ΔNx mutants display a much higher ratio of dyads: tetrads produced than WT cells, indicating an inability to complete both the reductional (MI) and equational (MII) divisions. This is also consistent with the phenotype observed for rfa2‐ΔNx mutants in mitotic cells, where checkpoint exit is affected by the presence and phosphorylation state of the Rfa2 N‐terminus.

Support or Funding Information

This research is supported by the award NSF‐CAREER‐1253723 to SJH.