Genetic regulation of genome stability in yeast

摘要 将正常细胞转化为癌细胞需要多种遗传变化。和几 例外情况，引发遗传不稳定性的突变或条件（例如，DNA复制应激）， 肿瘤的形成尚不清楚。该研究的目的是利用酵母菌 酿酒酵母作为一个模型，调查机制，产生各种类型的遗传不稳定性 与致癌有关。作为这种方法成功的一个例子，我们证明了DNA 酵母中的错配修复突变产生的微卫星不稳定性与遗传性 大肠癌细胞是一个重要的线索，作为因果突变。1.其中一项拟议的研究是 本发明的目的在于表征对碱基组合物有响应的新型增变子。我们发现，一个基因， GC含量高的基因突变率比GC含量低的基因高得多。我们随后发现 突变（met 18）使高GC基因的突变率提高了100倍以上， 对其他基因的影响Met 18蛋白是一种分子伴侣，参与将Fe-S簇转运到多种植物中。 参与DNA复制和DNA修复的酶。我们目前的数据表明，DNA聚合酶δ 缺少Fe-S簇的突变体具有显著降低的持续合成能力，这是met 18的突变体表型的原因。 这个假设将通过我们提出的实验来检验。2.着丝粒之间的融合通常是 在肿瘤细胞中观察到。我们已经开发了一个遗传系统， 发生在不同酵母染色体的着丝粒之间的事件。我们将使用该系统 研究着丝粒-着丝粒重组的遗传调控。3.我们最近使用了 哺乳动物APOBEC蛋白，它在单链DNA中脱氨基胞嘧啶，以绘制单链DNA的区域， 酵母细胞中的单链DNA经历DNA复制应激。使用APOBEC诱导的突变，我们 建议将我们的研究扩展到串联重复基因阵列中的缺失和重复。等 变异是基因组不稳定的重要原因。4.最后，Tel 1 p和Mec 1 p酵母蛋白是 分别与人ATM和ATR蛋白功能相关。两种人类基因的突变 与某些类型的肿瘤有关。具有tel 1 mec 1突变的酵母细胞具有显著升高的 染色体重排和染色体不分离。染色体重排，但不是 非整倍体是端粒融合率高的结果。我们在验证一个假设 染色体不分离水平是tel 1中组蛋白H2 A磷酸化缺陷的结果 mec 1菌株。

Abstract Multiple genetic changes are necessary to convert a normal cell into a cancer cell. With a few exceptions, the mutations or conditions (for example, DNA replication stress) that initiate genetic instability and tumor formation are not understood. The goal of the proposed research is to use the yeast Saccharomyces cerevisiae as a model for investigating the mechanisms that generate the various types of genetic instability related to carcinogenesis. As an example of the success of this approach, our demonstration that DNA mismatch repair mutations in yeast produced the same rate of microsatellite instability observed in hereditary colorectal cancer cells was an important clue as to the causal mutation. 1. One of the proposed studies is the characterization of a novel mutator that is responsive to base composition. We showed that a gene with high GC content had a much-elevated mutation rate than genes with lower GC contents. We subsequently identified a mutation (met18) that elevated the mutation rate of a high-GC gene more than 100-fold, with a much smaller effect on other genes. The Met18 protein is a chaperone involved in the transport of Fe-S clusters into a variety of enzymes involved in DNA replication and DNA repair. Our current data suggest that DNA polymerase delta lacking the Fe-S cluster has substantially reduced processivity, accounting for the mutator phenotype of met18. This hypothesis will be tested by our proposed experiments. 2. Fusions between centromeres are commonly observed in tumor cells. We have developed a genetic system that allows the identification of recombination events occurring between the centromeres of different yeast chromosomes. We will use this system to examine the genetic regulation of centromere-centromere recombination. 3. We have recently used the mammalian APOBEC protein, which deaminates cytosine in single-stranded DNA, to map regions of single- stranded DNA in yeast cells undergoing DNA replication stress. Using APOBEC-induced mutations, we propose extending our studies to map deletions and duplications in arrays of tandemly-repeated genes. Such alterations are an important cause of genome instability. 4. Lastly, the Tel1p and Mec1p yeast proteins are related functionally to the human ATM and ATR proteins, respectively. Mutations in both human genes are associated with certain classes of tumors. Yeast cells with tel1 mec1 mutations have greatly elevated rates of chromosome rearrangements and chromosome non-disjunction. The chromosome rearrangements, but not the aneuploidy, are a consequence of a high rate of telomere fusions. We are testing the hypothesis that the high level of chromosome non-disjunction is a consequence of defective phosphorylation of histone H2A in tel1 mec1 strains.