Deciphering the progression and regulation of human translesion DNA synthesis

项目总结/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 当它被共价修饰不断破坏时，它会复制，从而改变其编码特性。重点 是跨损伤DNA合成（TLS），主要的DNA损伤耐受性（DDT）的途径，利用在 复制受损的DNA TLS利用专门的DNA聚合酶， 一系列的DNA损伤，虽然降低的结晶度，并促进细胞存活，在面对DNA损伤， 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可以 选择性地繁殖具有增加的诱变和染色体重排的细胞，这可以导致 癌此外，由于TLS在暴露于DNA损伤剂后促进细胞存活，因此异常的细胞毒性可能是由细胞毒性引起的。 TLS还可以使癌细胞能够克服旨在引发细胞死亡的普通化疗 通过严重破坏DNA因此，TLS是靶向癌症联合治疗的有希望的候选者。尽管 尽管人类健康与TLS之间的联系已经建立，但TLS的进展和调控尚不清楚，许多 关键的差距仍然存在于我们的基础知识中，这些知识使我们对TLS的贡献的理解变得模糊， 遗传和致癌作用。例如，人类滴滴涕至少可以通过三种途径产生， TLS和其他DDT途径之间的相互作用是什么？我长期以来的兴趣和丰富的专业知识， 人类DNA损伤修复和复制以及我建立的利用多方面方法的能力 整合分子生物学、生物化学和生物物理学为填补这些空白提供了独特的机会。这 提案将通过解决两个广泛的领域来解决TLS的发展和监管问题， 我们对TLS和DDT的基本理解的基石; 1）TLS的激活; 2） 滴滴涕途径之间的相互作用。为此，我们设计并应用独特的定量方法， 动力学技术，可以适应许多生物场景。 PCNA滑动夹是一种重要的DNA复制因子，在DNA复制位点被单倍半胱氨酸化， Rad6（Rad18）2复合体的破坏。PCNA单泛素化对于人TLS是必要的，并激活 通过招募TLS因子来实现这一关键途径。第一区将研究人类的活动 Rad6（Rad18）2复合物被调节以在DNA损伤位点有效地单泛素化PCNA， TLS。人体DDT至少可以通过三种途径发生，包括TLS，所有途径都来自一个共同的 中间体区域2将研究TLS和其他DDT途径之间的相互作用， 每个通路中关键事件之间的功能关系。该提案在广泛的背景下研究TLS 它考虑了细胞环境的复杂性和动态性，并将大大推进我们的研究。 基本了解人类基因组如何在DNA损伤面前忠实地复制， 激发了癌症病因学和治疗研究的新途径。

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.