Structural and Mechanistic Studies of DNA Damage Bypass Pathways in Eukaryotes

项目摘要/摘要 DNA损伤是对基因组稳定性的严重威胁。这是因为它干扰了DNA 复制导致突变和染色体重排--这是癌症的特征， 衰老等疾病。为了确保基因组的稳定性，细胞利用DNA损伤旁路 应对复制过程中DNA损伤的途径。我们研究的长期目标是 程序是为了了解DNA损伤旁路是如何在真核系统中进行的 结构和机械层面。我们的研究将集中在两条损伤旁路上： 跨病变合成和模板切换。这一领域的进展最近有所放缓，因为 研究各种旁路组件如何组装成 并在大型的、动态的、多蛋白质复合体中发挥作用。我们已经开发了 需要克服的生化、生物、生物物理、计算和结构工具 这些挑战。这使我们处于一个独特的位置来回答许多基本问题 关于损害旁路的问题。我们未来的研究计划被组织成三个大的项目。首先，我们 将研究DNA损伤旁路的调控。这将通过确定如何绕过 复合体在停滞的复制分叉上组装，并通过确定此组装如何 由增殖细胞核抗原泛素化酶控制。第二，我们将研究其机制。 跨病变合成。这将通过确定最合适的非经典 选择聚合酶来绕过损伤，并通过确定每个非经典的 聚合酶适应受损的DNA模板。第三，我们将研究其机制。 模板切换。这将通过确定复制分叉的重塑如何完成 允许绕过DNA损伤，并通过确定这一过程是如何通过 叉形重塑DNA解旋酶。在回答这些问题时，我们将获得重要的新的 对维持基因组稳定性的洞察。

Project Summary/Abstract DNA damage is a serious threat to genome stability. This is because it interferes with DNA replication leading to mutations and chromosomal rearrangements – the hallmarks of cancer, aging, and other diseases. To ensure genome stability, cells utilize DNA damage bypass pathways to cope with DNA damage during replication. The long-term goal of our research program is to understand how DNA damage bypass is carried out in eukaryotic systems at the structural and mechanistic level. Our research will focus on two damage bypass pathways: translesion synthesis and template switching. Progress in this field has slowed recently because of the challenges associated with studying how the various bypass components assemble into and function within large, dynamic, multi-protein complexes. We have developed the biochemical, biological, biophysical, computational, and structural tools needed to overcome these challenges. This puts us in a unique position to answer many fundamental questions about damage bypass. Our future research plan is organized into three broad projects. First, we will study the regulation of DNA damage bypass. This will be done by determining how bypass complexes are assembled at stalled replication forks and by determining how this assembly is controlled by PCNA-ubiquitylating enzymes. Second, we will study the mechanisms of translesion synthesis. This will be done by determining how the most appropriate non-classical polymerase is chosen to bypass the damage and by determining how each non-classical polymerase accommodates damaged DNA templates. Third, we will study the mechanisms of template switching. This will be done by determining how the remodeling of the replication fork allows for the bypass of DNA damage and by determining how this process is carried out by fork-remodeling DNA helicases. In answering these questions, we will gain important new insights into the maintenance of genome stability.