| Summary: | 碩士 === 國立臺灣大學 === 生化科學研究所 === 101 === Programmed double-strand breaks (DSBs) are essential for a proper chromosome segregation during meiosis, because DSBs initiate physical connection between two homologous chromosomes through homologous recombination (HR). Dysegulation of DSBs formation can lead to the aneuploid of inviable gamete caused by abnormal chromosome disjunction. In the budding yeast, Saccharomyces cerevisiae, there are at least ten genes mediated the formation of DSBs including Mer2. The deletion of mer2 in yeast exhibits no DSB formation and poor meiotic spore. Recent study by Panizza et al. (2011) further infers that Mer2 protein accumulates on chromosome and recruits it’s interacting partners ; Mei4 and Rec114, to promote the formation of DSBs. However, it still remains largely unknown whether Mer2 itself possesses physical interaction with double-strand DNA and whether the DNA binding activity of Mer2 is prerequisite for generating DSBs. We aim to address this question by employing highly purified recombinant Mer2 protein for in vitro DNA binding analysis. Our results indicate that Mer2 is a DNA binding protein and possesses at least two DNA binding motifs. We further narrowed down the clusters of positive charge amino acids within Mer2 that contributes to its DNA binding ability, and currently in the progression of identifying those key residues. Besides identifying DNA binding defective mutant variants of Mer2, we will also examine whether those Mer2 mutants can rescue the phenotype of poor spore viability in the deletion of mer2 strain. We have successfully generated mer2 deletion in SK1 strain, and as reported, mer2 null showed poor spore viability. In the future, we will delineate whether Mer2 DNA-binding defective mutant variants can rescue the mer2 null phenotype by complementation experiments. Our findings will shield light on the mechanism of DNA binding property of Mer2 in contributing to make DNA double-strand breaks during meiosis.
|
|---|
