Mechanism of transcription-associated genome instability

项目摘要：基因组的变化，如突变、复制、缺失和 重组会带来癌症等躯体疾病，并推动进化过程。我的实验室已经 专注于了解这种基因组不稳定事件是如何以不一致的更高频率发生的 某些“热点”。与人们所熟知的将染色体描述为由 DNA、基因组更像是一条繁忙的高速公路，在那里许多蛋白质，包括拓扑异构酶，都在监测 不规则的螺旋扭转，结合和/或主动修饰DNA。此外，像RNA这样的蛋白质的巨型复合体 聚合酶和DNA聚合酶复合体动态移动、解离和强制扭曲 DNA在进行转录和复制时，有时会发生物理碰撞。为了 解释为什么突变/重组热点通常位于活跃的转录区域，我们查看了 多个涉及DNA的过程作为一个交互系统而不是作为每个独立的活动和 确定了基因组不稳定的转录相关原因。我的工作有助于证明这一点 由非正规性残基尿嘧啶和核糖核苷酸引起的突变在 转录激活。随后，我实验室的新发现导致了非复制型DNA的模型 发生在细胞周期的G1和G2期的合成导致活跃的尿嘧啶浓度较高 转录的基因。我们还发现了转录产生的负扭转应力与 这种升高的重组与DNA二级结构G-四链或G4DNA相关。我们进一步 确定抑制或加剧这种G4DNA诱导的基因组的G4DNA结合蛋白 不稳定。我的研究计划的中心目标是揭示基本和保守的机制 潜在的突变和基因组重排，这对细胞 转化为癌症与化疗药物的反应。根据我们之前的发现，我们将 继续解决重要的遗留问题：(1)使用易于处理的遗传方法进行研究 简单真核模式生物酵母中转录相关基因组的不稳定性 (2)开发创新的方法来测试尿嘧啶/核糖核苷酸掺入 DNA在G1和G2期间，以及(3)定义关键的G4 DNA结合之间的功能和结构相互作用 体外和体内的蛋白质和G4DNA。 我们正在进行的研究应该会进一步了解转录、复制和DNA 修复工作是为了基因组完整性的好处，也是为了损害基因组完整性。拥有一个全面的 基因组上发生的这些相互关联的动态过程的图像将有助于预测如何 抑制和纠正不利于正常细胞功能的基因组不稳定事件。

PROJECT SUMMARY: Changes in the genome such as mutagenesis, duplication, deletions, and recombination bring about somatic diseases like cancer and drive evolutionary processes. My lab has been focused on understanding how such genome instability events occur at incongruently higher frequencies at certain “hotspots.” Quite different from the familiar depiction of chromosomes as stationary strings made up of DNA, genome is more like a busy highway, where many proteins, including topoisomerases in surveillance for irregular helical torsion, bind and/or actively modify DNA. Moreover, mega-complexes of proteins like RNA polymerase and DNA polymerase complexes are dynamically moving along, unwinding, and forcibly distorting DNA while carrying out transcription and replication, sometimes physically colliding with each other. In order to explain why mutation/recombination hotspots are often located within actively transcribed regions, we viewed the multiple DNA-involving processes as an interactive system rather than as each independent activity and identified transcription-associated causes of genome instability. My work was instrumental in showing that mutations resulting from the non-canonical residues, uracil and ribonucleotide, are highly elevated upon transcription activation. Subsequently, novel discoveries in my lab led to the model that non-replicative DNA synthesis occurring in G1- and G2-phases of the cell cycles results in higher uracil density in actively transcribed genes. We also made key findings linking the transcription-generated negative torsional stress with the elevated recombination associated with the DNA secondary structure G-quadruplex or G4 DNA. We further identified G4 DNA-binding proteins that either suppress or exacerbate such G4 DNA-induced genome instability. The central goal of my research program is to uncover fundamental and conserved mechanism underlying mutagenesis and genome rearrangements, which will be important for both the cellular transformation into cancers and responses to chemotherapeutics. Building upon our previous findings, we will continue to address important remaining questions by (1) using tractable genetic approaches to study transcription-associated genome instability in the simple eukaryotic model organism Saccharomyces cerevisiae, (2) developing innovative approaches to test the model of uracil/ribonucleotide incorporation into DNA during G1 and G2, and (3) defining the functional and structural interaction between key G4 DNA-binding proteins and G4 DNA both in vitro and in vivo. Our ongoing investigation should further the understanding of how transcription, replication, and DNA repair work in conjunction either for the benefit or the detriment of genome integrity. Having a comprehensive picture of these interconnected and dynamic processes occurring on the genome will help in predicting how to suppress and correct genome instability events adverse to normal cellular functions.