Genetic and biochemical analysis of Drosophila MEI-9

果蝇 MEI-9 的遗传和生化分析

• 批准号: 6551673
• 负责人: HUTTON M KEARNEY
• 金额: $ 3.83万
• 依托单位: UNIV OF NORTH CAROLINA CHAPEL HILL
• 依托单位国家: 美国
• 财政年份: 2002
• 资助国家: 美国
• 起止时间: 2002-07-01 至 2003-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6551673
• 关键词: DNA repair; Drosophilidae; arthropod genetics; chemical stability; chromosome movement; endonuclease; gene mutation; genetic crossing over; meiosis; postdoctoral investigator

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.

未能解析减数分裂中间体作为交换导致高水平的染色体不分离，导致不育或不存活的后代。许多分子的细节已被描述为减数分裂重组的早期事件，但决议重组中间转换（或非转换）的产品仍有待确定。果蝇mei-9中的突变导致减数分裂交换的急剧减少和重组中间体中错配DNA的修复失败。mei-9编码核苷酸切除修复（NER）所需的核酸酶，并且mei-9的人类同源物XPF中的突变与癌症易感性相关。虽然MEI-9内切核酸酶在NER中的作用已被充分理解，但其在减数分裂中的功能仍不清楚。本文描述了减数分裂霍利迪连接被MEI-9内切核酸酶切割并且这种切割对于作为交换的有效解析是必需的建议。此外，它表明，引入的缺口MEI-9依赖性切割霍利迪路口作为一个切入点的核酸外切酶，以实现修复减数分裂中间体中的错配。这个模型允许人们对重组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mei-9文件的减数分裂染色体进行分子分析。此外，将在体外分析纯化的MEI-9蛋白（和相关蛋白）切割霍利迪连接的能力。