Mechanism of transcription-associated genome instability

项目总结：基因组的变化，如诱变，复制，缺失， 基因重组会导致癌症等躯体疾病，并推动进化过程。我的实验室 专注于了解这种基因组不稳定性事件是如何以不一致的更高频率发生的， 一些“热点”。与我们熟悉的将染色体描述为由 DNA、基因组更像是一条忙碌高速公路，许多蛋白质，包括拓扑异构酶，都在监视着 不规则螺旋扭转结合和/或主动修饰DNA。此外，像RNA这样的蛋白质的巨型复合物 聚合酶和DNA聚合酶复合体动态地沿着移动、解旋和强制扭曲 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，我们认为 多个涉及DNA的过程是一个相互作用的系统，而不是每个独立的活动， 确定了基因组不稳定性的转录相关原因。我的工作证明了 由非典型残基，尿嘧啶和核糖核苷酸引起的突变， 转录激活随后，我实验室的新发现导致了非复制性DNA 在细胞周期的G1期和G2期发生的尿嘧啶合成导致在细胞周期的G1期和G2期的尿嘧啶合成中， 转录基因我们还取得了关键的发现，将转录产生的负扭转应力与 与DNA二级结构G-四链体或G4 DNA相关的重组增加。我们进一步 鉴定了抑制或加剧这种G4 DNA诱导的基因组的G4 DNA结合蛋白 不稳定我的研究计划的中心目标是揭示基本的和保守的机制 潜在的诱变和基因组重排，这将是重要的细胞 转化为癌症和对化疗药物的反应。根据我们以前的发现，我们将 继续解决重要的剩余问题，通过（1）使用易处理的遗传方法来研究 简单真核模式生物酵母中与转录相关的基因组不稳定性 酿酒酵母，（2）开发创新的方法来测试尿嘧啶/核糖核苷酸掺入模型， 在G1和G2期间的DNA，和（3）定义关键G4 DNA结合之间的功能和结构相互作用 蛋白质和G4 DNA在体外和体内。 我们正在进行的研究应该进一步了解转录、复制和DNA 修复工作结合在一起，无论是对基因组的完整性的好处或损害。采取综合 这些发生在基因组上的相互关联和动态过程的图片将有助于预测如何 抑制和纠正对正常细胞功能不利的基因组不稳定性事件。

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.