Structural Investigations Critical to Understanding DNA Damage Recognition and Repair in Cancer

摘要 确保基因组的完整性对人类的健康、发展以及预防众多 疾病状态包括过早衰老和癌症。人类基因组不断受到破坏的威胁。 通过外源(紫外线，香烟烟雾中的化学物质)和内源(所需氧气 正常细胞呼吸)来源。虽然细胞氧化还原系统存在着用来解毒的 在新陈代谢的副产品中，有一部分不可避免地逃逸到与DNA接触并产生 氧化损伤，可通过自由基对碱基、链的破坏而导致复制过程中的突变 破发和基地损失的地点。据估计，每个细胞遭受的损伤部位超过10,000个 每天，如果不加以修复，就会导致疾病的形成并促进其进展。了解 识别和修复或绕过这些损坏部位的手段对于提供 关于癌症患者的预测结果和治疗策略的关键信息。这项建议 利用结构生物学技术，包括X射线结晶学，支持NCI资助的项目 旨在阐明参与DNA修复的关键酶的分子细节。项目1的重点是 识别和清除氧化损伤的DNA糖基酶的结构和生化研究 尽管程序2研究了DNA聚合酶β的复制保真度的机制， 修复酶负责填补由糖基酶产生的核苷酸缺口的修复聚合酶节目3 研究特殊的dna聚合酶，特别是Pollθ，及其在遇到未修复的情况下的反应。 复制过程中的DNA。POLθ以容易出错和上调的方式处理dna双链断裂 POLQ基因的缺失与乳腺癌患者不良的临床预后密切相关。Polθ因此 成为放射增敏联合治疗的引人注目的药物靶点。我在这项提案中的角色将 对聚合酶和糖基酶结合的复合体进行结构和生化研究 造成DNA损伤。我还将描述在癌症患者中发现的这些酶的变体。 此外，我负责维护X光设备和管理X光设备，负责培训新的 并指导学生和博士后在这些NCI项目中的结构工作。总而言之，这 这项工作有望对DNA修复蛋白的分子机制产生重要的见解。这些 预计结果将产生积极影响，因为对酶分子的详细了解 机制加上对癌症变异的研究将增加我们对癌症易感性的理解 并优化治疗方案。

SUMMARY Insuring genomic integrity is fundamental to human health, development as well as the prevention of numerous disease states including premature aging and cancer. The human genome is under constant threat of damage through both exogenous (UV Rays, chemicals from cigarette smoke) and endogenous (the oxygen required for normal cellular respiration) sources. While cellular oxidation-reduction systems exist for detoxifying a number of the metabolic byproducts, some fraction inevitably escapes to come in contact with the DNA and generate oxidized lesions that can lead to mutations during replication through free radical damage to bases, strand breaks and sites of base loss. It is estimated that each cell experiences in excess of 10,000 sites of damage per day that, if left unrepaired, lead to disease establishment and promote its progression. Understanding the means by which these sites of damage are recognized and repaired or bypassed is essential to providing critical information regarding predictive outcomes and therapeutic strategies for cancer patients. This proposal utilizes structural biology techniques including X-ray crystallography in support of thee NCI-funded programs aimed at elucidation of the molecular details of key enzymes involved in DNA repair. The focus of project 1 is the structural and biochemical investigations of DNA glycosylases that recognize and remove oxidative lesions within the DNA whereas program 2 investigates the mechanisms of replication fidelity of DNA polymerase β, the repair polymerase responsible for filling nucleotide gaps generated by a glycosylase. Program 3 investigates specialized DNA polymerases, particularly pol θ, and its response to encountering unrepaired DNA during replication. Pol θ processes DNA double strand breaks in an error-prone manner and upregulation of the POLQ gene strongly correlates with poor clinical outcome in breast cancer patients. Pol θ has thus emerged as a compelling drug target for combination therapy of radiosensitization. My role in this proposal will be to perform structural and biochemical investigations of complexes of polymerases and glycosylases bound to DNA damage. I will also be characterizing variants of these enzymes identified in cancer patients. Additionally, I maintain the X-ray equipment and manage the X-ray facility, am responsible for training of new users and guide students and postdocs in their structural work within these NCI programs. Collectively, this work is expected to generate important insights into the molecular mechanisms of DNA repair proteins. These results are expected to have a positive impact because the detailed knowledge of enzyme molecular mechanisms coupled with studies of cancer variants will increase our understanding of cancer susceptibility and optimize treatment protocols.