Genetic and biochemical analysis of Drosophila MEI-9

如果不能将减数分裂中间产物分解为杂交产物，就会导致高水平的染色体不分离，从而导致不育或后代死亡。许多分子细节已经被描述为减数分裂重组的早期事件，但重组中间产物到交叉(或非交叉)产物的分辨仍有待定义。果蝇mei-9的突变导致减数分裂交叉显著减少，并无法修复重组中间产物中不匹配的DNA。MEI-9编码核苷酸切除修复(NER)所需的核酸酶，人类MEI-9、XPF同源物的突变与癌症易感性有关。虽然Mei-9内切酶在NER中的作用已被很好地了解，但其在减数分裂中的功能仍不清楚。这里描述的是减数分裂Holliday连接被Mei-9内切酶切割的建议，并且这种切割是作为交换的有效拆分所必需的。此外，我们认为，依赖于Mei-9的Holliday连接的裂解引入的NICK是核酸外切酶修复减数分裂中间产物错配的切入点。这个模型使人们能够对重组DNA在减数分裂染色体上的排列做出可检验的预测。将对来自野生型和梅-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(和相关蛋白)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.