Regulation of mitotic genome stability in yeast.

酵母有丝分裂基因组稳定性的调节。

• 批准号: 10205748
• 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
• 财政年份: 2016
• 资助国家: 美国
• 起止时间: 2016-05-01 至 2026-04-30
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10205748
• 关键词: Affect; Chromosomes; Cryptococcus; DNA; DNA Repair; DNA Transposable Elements; DNA lesion; DNA strand break; Development; Environment; Enzymes; Evolution; Excision; Future; Genetic; Genetic Models; Genetic Recombination; Genetic Transcription; Genome; Genome Stability; Genomic Instability; Human; Ligation; Link; Loss of Heterozygosity; Mitotic; Modeling; Molecular; Movement; Mutagenesis; Nonhomologous DNA End Joining; Pathogenesis; Pathway interactions; Pharmaceutical Preparations; Process; Proteins; Regulation; Research; Ribonucleotides; Saccharomyces cerevisiae; Saccharomycetales; Site; Structure; System; Temperature; Topoisomerase; Yeasts; comparative; endonuclease; genome integrity; homologous recombination; human disease; interest; mutant; mutation assay; neoplastic cell; pathogenic fungus; repaired; tumor progression

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.

低水平的遗传不稳定性是适应和进化所必需的，但这种不稳定性也是一种强有力的驱动力。 人类疾病。我实验室的研究重点是遗传鉴定和分子表征， 过程，有助于有丝分裂基因组的不稳定性，以及DNA修复过程，促进基因组 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的完整性。双链断裂（DSB） 是最有害的DNA损伤之一，可以通过同源重组（HR）修复， 其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），其直接重新连接 破碎的结局。虽然两者都是固有的高保真过程，但HR可导致杂合性丢失，或 使分散的重复序列产生基因组重排。在NHEJ的情况下， 连接前的加工在连接不同DSB末端时在连接处产生小规模的变化 产生基因组重排。具有不同末端极性的内切核酸酶产生的DSB将被 用于在不同染色体上的序列趋异底物之间启动HR。比较分析 HR产物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。将检查在启动DSB的站点处的大序列不连续性的影响。 除了使用序列特异性酶来产生靶向DSB外，拓扑异构酶还可以断裂和重新连接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 与切口一端的共价连接;用化疗药物稳定切割中间体 到持续的高毒性断裂。我们先前描述了Top1的短缺失特征（I型 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II型酶，切割两条链产生DSB）启动了从头复制的形成 通过NHEJ途径。我们将研究如何从DNA中去除蛋白质的机制结束，以及如何 嵌入DNA中核糖核苷酸的存在影响Top2依赖性诱变。类似的重复是 在肿瘤细胞中发现了突变形式的TOP 2a，这种突变蛋白将在酵母中建模。基础上 我们长期致力于芽殖酵母实验系统中的重组和诱变， 扩大研究范围，包括人类真菌病原体隐球菌的诱变。 当隐球菌从环境转移到人类时，它必须迅速适应恶劣的条件 耐热性是致病的关键。使用正向突变分析，我们发现温度 模拟环境-人类转变的转变与转座因子的移动有关 （TE）。未来的研究将侧重于对温度依赖性TE运动的更全面分析， 动员的分子机制。