Polymerase Switching During Translesion DNA Synthesis within the Human System

描述（由申请人提供）：基因组DNA包含的信息必须被忠实地保存和精确地解码，以便遗传指令被准确地传递，细胞成分被正确地构建。尽管DNA非常稳定，但它仍然容易受到来自内源性和外源性来源的自发损伤。尽管DNA修复途径提供了保护，但一些损伤可能逃避检测并持续到s期。然而，复制聚合酶（pol）具有非常严格的聚合酶结构域和独特的“校对”结构域，以确保基因组DNA被准确复制。因此，对DNA碱基的破坏性修饰，统称为碱基损伤，会阻止或完全阻断复制叉的进展，导致其崩溃。重新启动失败常常导致双链断裂，这可能导致染色体重排、细胞周期阻滞和细胞死亡。因此，绕过这种复制阻滞并推迟对冒犯性损伤的修复以完成细胞周期并维持细胞存活通常是更有利的。这种任务可以通过翻译DNA合成（TLS）来完成，这是一种独特的过程，通过这种过程，DNA被复制过去的损伤，而不需要专门的TLS极点来修复它。由于具有更“开放”的聚合酶活性位点和缺乏校对活性，TLS poll能够以相对无错误的方式稳定地将dntp与受损模板相反，从而允许复制继续进行。然而，大约7个人类TLS poll中的每一个都以不同的准确度复制每个碱基病变。因此，为特定病变选择不合适的TLS pol可能导致受损DNA的错误复制。因此，必须严格规范TLS，以尽量减少复制错误。如果不这样做，可能会导致突变的积累，最终导致癌症。我们的长期目标是了解控制人类细胞中高效TLS的机制，以期识别促进突变和导致癌症发病的功能障碍。为了实现这一目标，我们已经开始研究在DNA复制过程中人类复制点和TLS点是如何交换的。在遇到损坏的DNA时，必须将复制DNA pol切换为TLS pol，以便继续复制。为了限制不太严格的TLS轮询的输入，并恢复TLS之后的高保真复制，必须反转此开关。使用集合和单分子动力学方法，包括FRET和最先进的零模式波导技术来可视化生物相关浓度的单分子，我们将在体外监测这些开关事件，以确定它们是如何协调的，以及如何在给定的病变中选择合适的TLS pol来实现有效的TLS。本提案的目标是确定在TLS期间如何控制端口切换，以及该控制如何确保有效和特定的端口切换以限制复制错误。这些知识将有助于识别导致TLS期间错误的pol切换的故障，并通过促进突变导致癌症的发生。

DESCRIPTION (provided by applicant): Genomic DNA contains information which must be faithfully maintained and precisely decoded in order for hereditary instructions to be accurately passed on and cellular components to be properly constructed. Although DNA is remarkably stable, it is nevertheless susceptible to spontaneous damage from endogenous and exogenous sources. Despite the protection provided by DNA repair pathways, some damage may evade detection and persist into S-phase. However, replicative polymerases (pols) have very stringent polymerase domains and distinct "proofreading" domains to ensure that genomic DNA is accurately copied. Consequently, damaging modifications to DNA bases, collectively referred to as base lesions, stall or completely block progression of the replication fork, causing it to collapse. Failure to restart often results in double-strand breaks which may lead to gross chromosomal rearrangements, cell-cycle arrest, and cell death. Therefore, it is often more advantageous to bypass such replicative arrests and postpone repair of the offending damage to complete the cell cycle and maintain cell survival. Such a task may be carried out by translesion DNA synthesis (TLS), a unique process by which DNA is replicated past damage without repairing it by specialized TLS pols. Characterized by a more "open" polymerase active site and the lack of proofreading activity, TLS pols are able to stably incorporate dNTPs opposite damaged templates in a relatively error-free manner, allowing replication to proceed. However, each of the 7 or so human TLS pols replicates each base lesion with varying levels of accuracy. Therefore, selection of the inappropriate TLS pol for a given lesion may result in erroneous replication of the damaged DNA. Thus, TLS must be tightly regulated to minimize replication errors. Failure to do so may lead to the buildup of mutations and ultimately cancer. Our long term goal is to understand the mechanisms that control efficient TLS in human cells in the hopes of identifying malfunctions which promote mutation and contribute to the onset of cancer. Towards this aim, we have begun to investigate how human replicative and TLS pols exchange during DNA replication. Upon encountering damaged DNA, a replicative DNA pol must be switched out for a TLS pol in order for replication to proceed. To limit the input of less-stringent TLS pols and resume high-fidelity replication following TLS, this switch must then be reversed. Using ensemble and single-molecule kinetic approaches, including FRET and state-of-the-art zero mode waveguide technology for visualizing single molecules at biologically relevant concentrations, we will monitor these switching events in vitro to determine how they are coordinated and how the appropriate TLS pol is selected for efficient TLS across a given lesion. It is the goal of this proposal to determine how pol switching is controlled during TLS and how this control ensures efficient and specific pol switching to limit replication errors. Such knowledge will aid in identifying malfunctions which lead to erroneous pol switching during TLS and contribute to the onset of cancer by promoting mutation. PUBLIC HEALTH RELEVANCE: The prevalence of cancer is astounding, and, according to the National Cancer Institute, approximately forty percent of all men and women in the United States alone will develop cancer during their lifetimes. Furthermore, translesion DNA synthesis often affords cancer cells the ability to become resistant to many commonly used chemotherapeutic agents. By elucidating the mechanisms underlying translesion DNA synthesis, this research will enhance our understanding of the causes of cancer and may ultimately lead to the development of novel methods of prevention and treatment, both of which are critical to eradicating this disease and vitally important to public health.