Genetic and biochemical analysis of Drosophila MEI-9

作为交叉的减数分裂中间体不能分离导致高水平的染色体不分离，导致不育或后代不能存活。许多分子细节已经描述了减数分裂重组的早期事件，但重组中间体进入交叉（或非交叉）产物的分辨率仍有待确定。果蝇mei-9的突变导致减数分裂交叉的急剧减少和重组中间体中不匹配DNA的修复失败。mei-9编码核苷酸切除修复（NER）所需的核酸酶，而mei-9的人类同源物XPF的突变与癌症易感性有关。虽然MEI-9内切酶在NER中的作用已被充分了解，但其在减数分裂中的功能仍不清楚。本文提出，减数分裂假日连接是由MEI-9内切酶切割的，这种切割是作为交叉的有效分辨率所必需的。此外，研究人员认为，mei -9依赖性的Holliday连接切割所引入的切口可以作为外切酶在减数分裂中间体中修复错配的切入点。这个模型允许人们对重组DNA在减数分裂染色体上的排列作出可检验的预测。将对野生型和mei-9文件的减数分裂染色体进行分子分析。此外，纯化的MEI-9蛋白（及相关蛋白）将在体外分析其切割Holliday连接的能力。

The failure to resolve meiotic intermediates as crossovers results in high levels of chromosome non-disjunction leading to sterility or inviable progeny. Many of the molecular details have been described for early events in meiotic recombination, but resolution of recombination intermediates into crossover (or non-crossover) products remains to be defined. Mutations in Drosophila mei-9 result in a dramatic decrease in meiotic crossovers and failure to repair mismatched DNA in recombination intermediates. mei-9 encodes a nuclease required for nucleotide excision repair (NER), and mutations in the human homologue of mei-9, XPF are associated with cancer predisposition. While the role of the MEI-9 endonuclease in NER is well understood, its function in meiosis remains unclear. Described herein is the proposal that meiotic Holliday junctions are cleaved by the MEI-9 endonuclease and that this cleavage is necessary for efficient resolution as a crossover. Additionally, it is suggested that the nicks introduced by MEI-9-dependent cleavage of Holliday junctions serve as an entry point for exonucleases to effect repair of mismatches in meiotic intermediates. This model allows one to make testable predictions about the arrangement of recombinant DNA on meiotic chromosomes. Molecular analysis will be performed on meiotic chromosomes derived from wild-type and mei-9 files. In addition, purified MEI-9 protein (and associated proteins) will be analyzed in vitro for the ability to cleave Holliday junctions.