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Regulation of mitotic genome stability in yeast.

酵母有丝分裂基因组稳定性的调节。

基本信息

批准号：
10613970
负责人：
SUE JINKS-ROBERTSON
金额：
$ 59.12万
依托单位：
DUKE UNIVERSITY
依托单位国家：
美国
项目类别：
财政年份：
2016
资助国家：
美国
起止时间：
2016-05-01 至 2023-06-30
项目状态：
已结题

项目摘要

A low level of genetic instability is required for adaptation and evolution, but such instability is also a potent driver of human disease. Research in my lab focuses on genetic identification and molecular characterization of processes that contribute to mitotic genome instability as well as DNA repair processes that promote genome stability. The proposed research will primarily use budding yeast (Saccharomyces cerevisiae) as a model to explore the repair of DNA strand breaks and how this impacts genome integrity. Double-strand breaks (DSBs) are among the most detrimental of DNA lesions and are repaired either by homologous recombination (HR), which uses an intact duplex as a repair template, or by nonhomologous end joining (NHEJ), which directly rejoins broken ends. Although both are inherently high-fidelity processes, HR can result in loss of heterozygosity or can engage dispersed repeated sequences to generate genome rearrangements. In the case of NHEJ, end processing prior to ligation produces small-scale changes at the junction while joining the ends of different DSBs generates genome rearrangements. Endonuclease-generated DSBs that have different end polarities will be used to initiate HR between sequence-diverged substrates on different chromosomes. Comparative analyses of HR product types and their strand compositions will reveal how end structure affects mitotic HR intermediates and mechansims. The effects of large sequence discontinuities at the site of an initiating DSB will be examined. In addition to use of sequence-specific enzymes to create targeted DSBs, topoisomerases break and rejoin DNA strands to resolve topological problems that arise during transcription and replication. These enzymes form a covalent link with one end of a nick; stabilization of cleavage intermediates with chemotherapeutic drugs leads to persistent breaks that are highly toxic. We previously described a short-deletion signature of Top1 (a type I enzyme that nicks one DNA strand) and defined the associated molecular mechanism. We recently discovered that Top2 (a type II enzyme that nicks both strands to create a DSB) initiates the formation of de novo duplications through the NHEJ pathway. We will examine how the mechanism of protein removal from DNA ends and how the presence of ribonucleotides embedded in DNA affect Top2-dependent mutagenesis. Similar duplications are found in tumor cells with a mutant form of TOP2a, and this mutant protein will be modeled in yeast. Building on our long-term interests in recombination and mutagenesis in the budding yeast experimental system, we recently expanded studies to include mutagenesis in the human fungal pathogen Cryptococcus deneoformans. Cryptococcus must rapidly adapt to hostile conditions when it transitions from the environment to the human host, and heat tolerance is critical for pathogenesis. Using a forward mutation assay, we found that a temperature shift mimicking the environment-human transition is associated with the mobilization of transposable elements (TEs). Futures studies will focus on a more global analysis of temperature-dependent TE movement and the molecular mechanism(s) of mobilization.

适应和进化需要低水平的遗传不稳定性，但这种不稳定性也是一个强有力的驱动因素的人类疾病。我实验室的研究重点是遗传鉴定和分子表征导致有丝分裂基因组不稳定的过程以及促进基因组的 DNA 修复过程稳定。拟议的研究将主要使用芽殖酵母（酿酒酵母）作为模型探索 DNA 链断裂的修复以及这如何影响基因组完整性。双链断裂 (DSB) 是最有害的 DNA 损伤之一，可通过同源重组 (HR) 进行修复，它使用完整的双链体作为修复模板，或通过非同源末端连接（NHEJ）直接重新连接断头。尽管两者本质上都是高保真度过程，但 HR 可能会导致杂合性丢失或可能参与分散的重复序列以产生基因组重排。对于 NHEJ，结束连接前的处理会在连接不同 DSB 末端时在连接处产生小范围的变化产生基因组重排。核酸内切酶生成的具有不同末端极性的 DSB 将被用于启动不同染色体上序列分歧底物之间的同源重组。比较分析 HR 产品类型及其链组成将揭示末端结构如何影响有丝分裂 HR 中间体和机制。将检查起始 DSB 位置处大序列不连续性的影响。除了使用序列特异性酶来创建靶向 DSB 之外，拓扑异构酶还可断裂并重新连接 DNA 链来解决转录和复制过程中出现的拓扑问题。这些酶形成与切口一端共价连接；用化疗药物先导稳定裂解中间体导致剧毒的持续断裂。我们之前描述了 Top1 的短删除签名（I 型一种在一条 DNA 链上产生切口的酶）并定义了相关的分子机制。我们最近发现 Top2（一种 II 型酶，可在两条链上形成切口以产生 DSB）启动从头重复的形成通过 NHEJ 途径。我们将研究从 DNA 中去除蛋白质的机制如何结束以及如何 DNA 中嵌入的核糖核苷酸的存在会影响 Top2 依赖性诱变。类似的重复是在具有 TOP2a 突变形式的肿瘤细胞中发现了这种突变蛋白，该突变蛋白将在酵母中进行建模。建立在我们对芽殖酵母实验系统中的重组和诱变的长期兴趣，我们最近扩大研究范围，包括人类真菌病原体隐球菌的诱变。当隐球菌从环境过渡到人类时，必须迅速适应恶劣的条件宿主，耐热性对于发病机制至关重要。使用正向突变测定，我们发现温度模仿环境-人类转变的转变与转座因子的动员有关（TE）。未来的研究将侧重于对温度相关的 TE 运动进行更全面的分析以及动员的分子机制。