Polymerase Switching During Translesion DNA Synthesis within the Human System

描述（由申请人提供）：基因组DNA包含必须忠实保存和精确解码的信息，以便准确传递遗传指令和正确构建细胞成分。虽然DNA非常稳定，但它仍然容易受到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，但一些损伤可能会逃避检测并持续到S期。然而，复制型聚合酶（pols）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，停止或完全阻止复制叉的进展，导致其崩溃。未能重新启动通常会导致双链断裂，这可能导致染色体严重重排、细胞周期停滞和细胞死亡。因此，它往往是更有利的绕过这种复制停滞和推迟修复的冒犯性损害，以完成细胞周期和维持细胞存活。这样的任务可以通过跨损伤DNA合成（TLS）来进行，这是一种独特的过程，通过该过程，DNA在损伤后复制而不通过专门的TLS pols修复。TLS pols的特征在于更“开放”的聚合酶活性位点和缺乏校对活性，TLS pols能够以相对无错误的方式稳定地将dNTP与受损的模板相对，从而允许复制进行。然而，7个左右的人类TLS pols中的每一个都以不同的准确度复制了每个基底病变。因此，对于给定病变选择不适当的TLS pol可能导致受损DNA的错误复制。因此，TLS必须严格监管，以最大限度地减少复制错误。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有效TLS的机制，以期识别促进突变并导致癌症发生的功能障碍。为了实现这一目标，我们已经开始研究人类复制和TLS pols如何在DNA复制过程中交换。在遇到损坏的DNA时，必须将复制DNA pol切换为TLS pol以进行复制。要限制不太严格的TLS pols的输入并在TLS之后恢复高保真复制，则必须反转此开关。使用合奏和单分子动力学的方法，包括FRET和国家的最先进的零模式波导技术可视化单分子在生物学相关的浓度，我们将监测这些开关事件在体外，以确定它们是如何协调，以及如何适当的TLS波尔选择有效的TLS在给定的病变。本提案的目标是确定在TLS期间如何控制pol切换，以及这种控制如何确保有效和特定的pol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，并通过促进突变而导致癌症的发生。

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.