Genome instability resulting in chromosome mis-segregation during meiosis accounts for as many as 50% of human miscarriages and contributes to birth defects such as Down Syndrome. Maintenance of genome stability during the normal cell cycle is closely tied to DNA replication fork stability and repair. This requires active checkpoint signaling pathways mediated by several highly conserved kinases. Studies in model organisms, especially yeasts, has provided critical insights into the mechanisms by which these kinases function in proliferating cells. However, there has been little investigation of how "normal" checkpoints function in meiotic S phase (meiS) in response to replication stress. Instead, most studies of meiosis have focused on key events downstream of DNA replication, including formation of programmed double strand breaks, recombination and the reductional meiosis I division. Thus, there is a critical gap in understanding how meiotic cells respond to replication stress and DNA damage during meiS phase, which is separate from the activation of the meiotic recombination program. Intriguingly, evidence suggests that the checkpoint response pathways in meiS phase are radically reprogrammed. Instead of preventing DNA damage, they actually promote it, and thus help create substrates for recombination, which is necessary for proper chromosome segregation. Indeed, the S phase checkpoint kinase Cds1 (ScRad53) and the replication kinase Hsk1 and its subunit Dfp1 (ScCdc7, ScDbf4) which normally preserve replication fork integrity and promote DNA repair, instead actively promote double strand break formation during meiosis. The parent proposal addresses how the cell adjusts its sensitivity to replication fork stability to facilitate the meiotic differentiation proram. This supplemental revision examines the molecular consequences of replication fork instability in meiosis by asking how fork collapse impacts the recruitment and distribution of meiosis-specific proteins relative to breaks, including the meiotic endonuclease Rec12Spo11, the meiotic cohesin Rec8, and components of the linear elements that link homologues together. We hypothesize that breaks induced by fork collapse will influence the distribution these protein, and thus impact the distribution of DNA breaks. This revision will deep sequencing technologies combined with chromatin immunoprecipitation (ChIP-seq) to query the genome and determine how replication stress impacts these macromolecular interactions during meiosis. While these methods are new to our laboratory, there is substantial local expertise that will help us bring thi on board to complement our cell biology approaches. As a result of these experiments, we will be able to correlate replication origins, fork collapse, and programmed double strand breaks under conditions of genome instability in meiosis. This will provide important insights into the stresses that impact meiotic progression in humans as well as yeast.
A substantial fraction of birth defects result from chromosomal defects in meiosis, the process that produces eggs and sperm. This project uses modern genomic techniques in a simple yeast to study how chromosomes in meiosis respond to stresses that may contribute to meiotic defects. The goal is to identify how cellular stresses particularly during DNA replication could contribute to birth defects.
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