MEI-9 FUNCTION IN DROSOPHILA MEIOTIC RECOMBINATION

基因重组是减数分裂中同源染色体准确分离的必要条件。许多减数分裂重组途径现在在分子水平上被理解，主要是由于对酿酒酵母的研究。然而，人们对这一途径中的最后事件仍然知之甚少，并且很少有蛋白质被确定为执行这些事件的候选蛋白。果蝇的mei-9基因在这些后期阶段起作用。MEI-9蛋白是核苷酸切除修复（NER）内切酶的一个组成部分，它可以切割具有特定极性的单链到双链转变的DNA结构。MEI-9的人类同源基因是XPF，这是一种与遗传性色素干皮病有关的基因，与皮肤癌有关。对于MEI-9的减数分裂功能，一个有吸引力的模型是它切断假日连接来分解重组中间体。本提案中的实验试图检验这一模型。野生型和mei-9突变体的减数分裂重组事件的半四分体（两条染色单体）将被表征，提供有关重组中间体结构及其分解方式的信息。MEI-9蛋白可以用抗MEI-9抗体或表位标签进行免疫定位。将对mei-9的15个突变等位基因进行分子和遗传学表征，以了解该蛋白的结构与功能之间的关系。mus210基因编码果蝇的第二个亚基。该基因的突变会破坏NER，但不会破坏减数分裂重组。因此，这些要么是功能分离等位基因，要么在减数分裂中不需要这种蛋白质的活性。如果后者是正确的，这是两个亚单位之间功能分离的第一个情况。为了验证这一点，新的mus210突变将被恢复并表征。如果MEI-9不需要MUS210，则可能是MUS210存在并二聚化，但不需要其活性，或者蛋白质存在但不与MEI-9二聚化，或者MUS210不存在于减数分裂细胞中。因此，将分析该蛋白的表达。此外，将强制表达该蛋白，以确定其是否干扰MEI-9的功能。最后，将确定MEI-9的减数分裂特异性伴侣。

Genetic recombination is essential for the accurate segregation of homologous chromosomes from one another in meiosis. Much of the meiotic recombination pathway is now understood at the molecular level, largely due to studies in S. cerevisiae. The final events in the pathway, however, remain very poorly understood, and few proteins have been identified as candidates for carrying out these events. The Drosophila mei-9 gene acts during these late stages. The MEI-9 protein is a component of a nucleotide excision repair (NER) endonuclease, which cuts DNA structures that undergo single-stranded to double-stranded transitions with a specific polarity. The human homolog of MEI-9 is XPF, a gene implicated in the hereditary disorder xeroderma pigmentosum, which is associate with skin cancer. An attractive model for the meiotic function of MEI-9 is that it cuts Holliday junctions to resolve recombination intermediates. Experiments in this proposal seek to test this model. Half-tetrads (two chromatids) of meiotic recombination events will be characterized molecular from both wild type and mei-9 mutants, providing information about the structure of the recombination intermediate and the manner in which it is resolved. MEI- 9 protein will be immunolocalized with anti-MEI-9 antibodies or with epitope tags. Fifteen mutant alleles of mei-9 will be characterized molecularly and genetically to learn about how the structure of the protein correlates with its function. The mus210 gene encodes the second subunit in Drosophila. Mutations in this gene disrupt NER, but not meiotic recombination. Hence, either these are separation-of-function alleles, or the activity of this protein is not required in meiosis. If the latter is true, this is the first case of separation of function between the two subunits. To test this, new mus210 mutations will be recovered and characterized. If MEI-9 does not require MUS210, it may be that MUS210 is present and dimerizes, but its activity is not required, or that the protein is present but does not dimerize with MEI-9, or that MUS210 is not present in meiotic cells. The expression of the protein will therefore be analyzed. In addition, expression of the protein will be forced, to determine whether it interferes with the MEI-9 function. Finally, potential meiosis-specific partners of MEI-9 will be identified.