Regulation of mitotic genome stability in yeast.

适应和进化需要低水平的遗传不稳定，但这种不稳定也是一个强有力的驱动因素。 人类疾病的威胁。我实验室的研究主要集中在基因鉴定和分子特征方面 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 稳定性。拟议的研究将主要使用萌芽酵母(酿酒酵母)作为模型，以 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，通过同源重组(HR)修复， 它使用完整的双链作为修复模板，或者通过非同源末端连接(NHEJ)直接重新连接 断头。虽然两者都是本质上高保真的过程，但HR可能会导致杂合性丧失或 利用分散的重复序列来产生基因组重排。在NHEJ的情况下，结束 在连接不同DSB的末端时，在连接之前的处理会在连接处产生小范围的变化 产生基因组重排。由核酸内切酶产生的具有不同末端极性的DSB将被 用于在不同染色体上序列分歧的底物之间启动HR。比较分析两种方法 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 和机械模拟。将检查在启动DSB的位置处的大序列不连续的影响。 除了使用序列特定的酶来产生靶向DSB外，拓扑异构酶还会断裂并重新连接DNA 链来解决转录和复制过程中出现的拓扑问题。这些酶形成一种 与缺口一端的共价连接；用化疗药物稳定裂解中间产物 到剧毒的持续性休息。我们之前描述了Top1(一种类型I)的短删除签名 切割一条DNA链的酶)并定义了相关的分子机制。我们最近发现 TOP2(一种切割两条链以产生DSB的II型酶)启动从头复制的形成 通过NHEJ途径。我们将研究从DNA中去除蛋白质的机制是如何结束的，以及如何 DNA中核糖核苷酸的存在会影响TOP2依赖的突变。类似的复制是 在带有TOP2A突变形式的肿瘤细胞中发现，这种突变蛋白将在酵母中建模。在基础上建设 我们对萌芽酵母实验系统中的重组和突变的长期兴趣，我们最近 扩大研究范围，包括人类真菌病原体脱氧隐球菌致突变。 当隐球菌从环境过渡到人类时，它必须迅速适应恶劣的条件 耐热性是致病的关键。使用正向突变测试，我们发现一个温度 模仿环境的转变-人类的转变与转座元件的动员有关 (TES)。未来的研究将集中在对依赖于温度的TE运动和 动员的分子机制(S)。

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.