Novel functions of PrimPol in ribonucleotide-induced genome instability

摘要 长期以来，基因组不稳定性一直被认为是癌症、神经退行性变和 衰老核糖核苷酸（rNMP）代表基因组DNA中最丰富的非规范核苷酸， 是基因组不稳定性的主要来源。了解rNMP如何整合到DNA中很重要 用于定义基因组不稳定性和疾病发病机理的基本机制。rNMP掺入 是DNA中rNMP污染的主要来源，然而， rNMP插入频率对于新的DNA聚合酶/引发酶（PrimPol）是未知的。知识缺口 存在于对PrimPol对DNA中rNMP污染的贡献的理解中， 其蛋白质相互作用伙伴的功能。PrimPol是一种新发现的多功能人类translesion TLS是一种DNA聚合酶/引发酶，与许多其他人类TLS pols有根本区别。 这是因为除了DNA损伤旁路，PrimPol还可以进行从头DNA/RNA合成， 与来源无关的重新启动。该项目的长期目标是阐明遗传机制， 不稳定性，确定参与的关键酶的机制，并开发新的策略，以减少 基因组不稳定性及其致病作用。本申请的目的是确定 PrimPol相对于其他几种重要的TLS Pol对DNA中rNMP掺入的贡献，以及 阐明其蛋白质相互作用伴侣，复制蛋白A（RPA）的调节功能。中央 假设PrimPol通过rNMP掺入导致基因组不稳定性，而RPA对此进行调节 活动这个假设将以两个目标进行检验。目标1，量化效率、频率和 使用稳态、预稳态动力学的PrimPol-catalyzed rNMP掺入的序列特异性 分析、基于LC-MS的寡核苷酸测序和计算机模拟。几种与癌症有关的 变体将表征其复制保真度、rNMP掺入频率和催化活性。 能力目的2，阐明RPA对PrimPol-DNA掺入的调控作用 使用稳态动力学分析。将开发一种新的竞争性测定法来评估 PrimPol的DNA聚合酶和引发酶活性。拟议的工作意义重大，因为它将 推进对基因组不稳定性和疾病发病机制的新机制的理解， 将为新疗法的发展提供信息。这项建议是创新的，因为它将首次， 定量确定PrimPol及其变体对DNA中rNMP污染的贡献，以及 确定RPA-PrimPol相互作用的生物学功能，这将推动我们目前的研究。 了解PrimPol的生物学功能及其在基因组不稳定性中的作用。拟议 研究计划将为学生提供有意义的生物医学培训机会，并大大提高 中密歇根大学的整体生物医学研究环境。

ABSTRACT Genome instability has long been implicated as a main causal factor in cancer, neurodegeneration and aging. Ribonucleotide (rNMP) represents the most abundant non-canonical nucleotide in genomic DNA and is a major source of genome instability. Understanding how rNMPs are incorporated into DNA is important for defining fundamental mechanisms of genome instability and disease pathogenesis. rNMP incorporation by DNA polymerases is a major source of rNMP contamination in DNA, however the efficiency and frequency of rNMP insertion are unknown for a novel DNA polymerase/primase (PrimPol). A knowledge gap exists in the understanding of the contribution of PrimPol to rNMP contamination in DNA and in the functions of its protein-interaction partners. PrimPol is a newly discovered, versatile human translesion synthesis (TLS) DNA polymerase/primase that is fundamentally distinct from many other human TLS pols. This is because in addition to DNA lesion bypass, PrimPol can perform de novo DNA/RNA synthesis and origin-independent re-priming. The long-term goal of the project is to elucidate the mechanisms of genetic instability, determine the mechanisms of key enzymes involved, and developing novel strategies to reduce genome instability and its pathogenic effects. The objective of this application is to determine the contribution of PrimPol to rNMP incorporation in DNA relative to several other important TLS pols, and to elucidate the regulatory functions of its protein interaction partner, replication protein A (RPA). The central hypothesis is that PrimPol contributes genome instability via rNMP incorporation and RPA regulates this activity. This hypothesis will be tested with two aims. Aim 1, to quantify the efficiency, frequency and sequence specificity of PrimPol-catalyzed rNMP incorporation using steady-state, pre-steady-state kinetic analyses, LC-MS based oligonucleotide sequencing and computer simulations. Several cancer-related variants will be characterized for its replication fidelity, rNMP incorporation frequency and catalytic competency. Aim 2, to elucidate the role of RPA in modulating PrimPol-catalyzed nucleotide incorporation using steady-state kinetic analysis. A novel competitive assay will be developed to assess the preference of PrimPol for its DNA polymerase and primase activities. The proposed work is significant because it will advance the understanding of novel mechanisms of genomic instability and disease pathogenesis, which will inform the development of new therapeutics. This proposal is innovative because it will, for the first time, quantitatively define the contribution of PrimPol and its variants to rNMP contamination in DNA, and determine the biological functions of RPA-PrimPol interactions, which will advance our current understanding of the biological function of PrimPol and its role in genomic instability. The proposed research program will offer meaningful biomedical training opportunities for students and greatly enhance the overall biomedical research environment at Central Michigan University.