Deciphering the progression and regulation of human translesion DNA synthesis

破译人类跨损伤 DNA 合成的进展和调节

• 批准号: 10669748
• 负责人: Mark Hedglin
• 金额: $ 39.43万
• 依托单位: PENNSYLVANIA STATE UNIV HERSHEY MED CTR
• 依托单位国家: 美国
• 财政年份: 2022
• 资助国家: 美国
• 起止时间: 2022-08-01 至 2027-05-31
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10669748
• 关键词: Acute; Area; Biochemistry; Biological; Biophysics; Cancer Etiology; Cell Death; Cell Survival; Cells; Chromosomal Rearrangement; Closure by clamp; Code; Complex; Critical Pathways; DNA Damage; DNA Repair; DNA Replication Damage; DNA Replication Factor; DNA biosynthesis; DNA-Directed DNA Polymerase; Disease; Environment; Event; Exposure to; Fibroblast Growth Factor; Functional disorder; Genetic; Genome; Goals; Health; Human; Human Activities; Human Genome; Kinetics; Knowledge; Link; Malignant Neoplasms; Modification; Molecular; Molecular Biology; Monoubiquitination; Mutagenesis; Pathway interactions; Property; Regulation; Research; Research Proposals; Site; Slide; Techniques; cancer cell; cancer therapy; carcinogenesis; chemotherapy; design; human DNA damage; interest; programs; recruit

Project Summary/Abstract The overarching goal of my research program is to understand how the human genome is faithfully replicated when it is constantly damaged by covalent modifications that can alter its coding properties. A focus is on translesion DNA synthesis (TLS), the predominant DNA damage tolerance (DDT) pathway utilized in humans to replicate damaged DNA. TLS utilizes specialized DNA polymerases that can accommodate an array of DNA damages, albeit with lowered fidelities, and promotes cell survival in the face of DNA damage by allowing replication of a damaged genome to continue. However, tight regulation is critical as aberrant TLS can selectively propagate cells with increased mutagenesis and chromosomal rearrangements, which can lead to cancer. Furthermore, because TLS promotes cell survival after exposure to DNA damaging agents, aberrant TLS can also afford cancer cells the ability to overcome common chemotherapies that aim to trigger cell death by acutely damaging DNA. Hence, TLS is a promising candidate for targeted cancer co-therapy. Despite the established links between human health and TLS, the progression and regulation of TLS is unclear and many key gaps persist in our fundamental knowledge that cloud our understanding of the contribution of TLS to genetic inheritance and carcinogenesis. For example, human DDT can occur by at least three pathways but what is the interplay between TLS and other DDT pathways? My long-standing interests and vast expertise in human DNA damage repair and replication and my established ability to utilize a multi-faceted approach integrating molecular biology, biochemistry, and biophysics provide a unique opportunity to fill these gaps. This proposal will address the progression and regulation of TLS by tackling two broad areas that are each a cornerstone of our fundamental understanding of TLS and DDT in general; 1) Activation of TLS and; 2) The interplay between DDT pathways. To do so, we design and apply unique, quantitative approaches that utilize kinetic techniques and can be adapted to many biological scenarios. The PCNA sliding clamp is an essential DNA replication factor and is monoubiquitinated at sites of DNA damage by the Rad6(Rad18)2 complex. PCNA monoubiquitination is imperative for human TLS and activates this critical pathway by recruiting TLS factors. Area 1 will investigate how the activity of the human Rad6(Rad18)2 complex is regulated to efficiently monoubiquitinate PCNA at DNA damage sites, activating TLS. Human DDT can occur by at least three pathways including TLS and all emanate from a common intermediate. Area 2 will investigate the interplay between TLS and other DDT pathways and decipher functional relationships between key events in each pathway. This proposal investigates TLS in broad contexts that consider the complexities and dynamics of cellular environments and will significantly advance our fundamental understanding of how the human genome is faithfully replicated in the face of DNA damage and inspire new avenues of research in cancer etiology and treatment.

项目摘要/摘要 我的研究计划的首要目标是了解人类基因组是如何忠实地 当它被可能改变其编码性质的共价修饰不断破坏时复制。一个焦点 是关于跨损伤DNA合成(TLS)，这是主要的DNA损伤耐受(DDT)途径，在 人类复制受损的DNA。TLS利用专门的DNA聚合酶，可以适应 一系列DNA损伤，尽管保真度较低，并通过以下方式促进细胞存活 使受损基因组的复制得以继续。然而，严格的监管是至关重要的，因为异常的TLS可能会 选择性地繁殖具有更多突变和染色体重排的细胞，这可能导致 癌症。此外，由于TLS在暴露于DNA损伤剂后促进细胞存活，异常 TLS还可以为癌细胞提供克服旨在引发细胞死亡的常见化疗药物的能力 通过严重破坏DNA。因此，TLS是靶向癌症联合治疗的一个很有前途的候选者。尽管 在人类健康和TLS之间建立了联系，TLS的进展和调控尚不清楚，有许多 我们的基础知识中仍然存在着关键的差距，这些差距影响了我们对TLS对 遗传和致癌。例如，人类滴滴涕可以通过至少三种途径发生，但 TLS和其他滴滴涕途径之间的相互作用是什么？我的长期兴趣和丰富的专业知识 人类DNA损伤修复和复制，以及我利用多方面方法的既定能力 分子生物学、生物化学和生物物理学的结合为填补这些空白提供了独特的机会。这 提案将通过处理两个广泛的领域来解决TLS的进展和监管问题，这两个领域分别是 我们对TLS和DDT的基本理解的基石；1)TLS的激活；2)TLS和 滴滴涕途径之间的相互作用。为了做到这一点，我们设计并应用了独特的量化方法，利用 动力学技术，可适用于许多生物学场景。 增殖细胞核抗原滑动夹是一种重要的DNA复制因子，在DNA的位点上是单核苷酸的 受Rad6(Rad18)2络合物的破坏。增殖细胞核抗原单素化是人类TLS的必由之路，并激活 这一关键途径是通过招募TLS因子实现的。第一区将调查人类的活动是如何 Rad6(Rad18)2复合体被调节为在DNA损伤部位高效地单核苷酸激活增殖细胞核抗原，激活 TLS。人类滴滴涕至少可以通过三种途径发生，包括TLS，并且都来自共同的 中级的。区域2将调查TLS与其他滴滴涕途径之间的相互作用并破译 每条通路中关键事件之间的功能关系。本提案在广泛的背景下对TLS进行调查 考虑到蜂窝环境的复杂性和动态性，并将显著推动我们的 基本了解人类基因组是如何在DNA损伤和 启发癌症病因学和治疗研究的新途径。