Genetic regulation of genome stability in yeast

摘要 将正常细胞转化为癌细胞需要多种基因改变。只有几个 例外情况，引起遗传不稳定和 肿瘤的形成还不清楚。这项拟议的研究的目标是使用酵母 酿酒酵母作为研究各种类型遗传不稳定性产生机制的模型 与致癌有关。作为这种方法成功的一个例子，我们展示了DNA 酵母错配修复突变产生的微卫星不稳定性与遗传性突变相同 结直肠癌细胞是这种原因突变的重要线索。1.其中一项建议的研究是 一种对碱基组成有反应的新型突变子的特征。我们证明了一种高密度的基因 GC含量较低的基因突变率明显高于GC含量较低的基因。我们随后确定了 一种突变(Met18)，将高GC基因的突变率提高100倍以上，而突变的幅度要小得多 对其他基因的影响。Met18蛋白是一种伴侣蛋白，参与了铁-S簇向多种物种的运输 参与DNA复制和DNA修复的酶。我们目前的数据表明DNA聚合酶增量 缺乏Fe-S簇大大降低了加工能力，这是met18突变子表型的原因。 这一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。我们已经开发了一种基因系统，可以识别重组 发生在不同酵母染色体着丝粒之间的事件。我们将使用这个系统来 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，它能使单链DNA中的胞嘧啶脱氨基，以定位单链DNA的区域 在经历DNA复制压力的酵母细胞中搁置的DNA。利用APOBEC诱导的突变，我们 建议将我们的研究扩展到定位重复重复基因阵列中的缺失和复制。是这样的 突变是基因组不稳定的一个重要原因。4.最后，Tel1p和Mec1p酵母蛋白是 分别与人类ATM和ATR蛋白在功能上相关。人类两种基因的突变都是 与某些类型的肿瘤有关。带有tel1 Mec1突变的酵母细胞大大提高了 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。我们正在检验这样一种假设，即 染色体不分离水平是TEL1中组蛋白H_2A磷酸化缺陷的结果 Mec1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.