Mechanisms of DNA replication and maintenance in eukaryotes

描述：本提案的重点是真核生物 DNA 复制机制的研究 细胞 (1) 以及复制功能障碍对自发性和损伤的影响- 诱导突变 (2)，以及细胞周期检查点激活 (3)。我们的主要方法结合 对酿酒酵母进行生化和生物物理分析以及遗传分析，以获得 深入了解这三个广泛定义的途径及其相互关联性。我们的研究是 通过对在这些过程中发挥作用的复杂机械的结构研究进行增强。提议 DNA 复制研究基于滞后链机制定义方面的大量进展记录 DNA 复制和冈崎片段成熟。由于冈崎碎片代表了迄今为止最 所有细胞中都频繁出现 DNA 不连续性，因此有必要了解 DNA 的不同层次的调控 这个过程。冈崎片段成熟主要在明确的生化系统中进行研究， 与复制叉处发生的其他事件隔离。我们建议在以下范围内扩大我们的研究 完整的复制叉的背景，它已在酵母复制起点处组装。我们的 诱变研究将集中在主要参与者 DNA 聚合酶 z 和 Rev1 上。一方面，Rev1 促进 Pol z 绕过 DNA 损伤；另一方面，它通过抑制 Pol 来限制诱变的程度 病变狭窄环境外的 z 依赖性 DNA 合成。我们将揭开机制 这是 Rev1 双重调节功能的基础。我们检查点研究的主要重点是 两种传感器蛋白激酶 Mec1 和 Tel1，分别是人类 ATR 和 ATM 的直系同源物。 生化和遗传学研究将与冷冻电镜研究相结合，以了解基础如何 这些独特的蛋白激酶的活性被激活。此外，拥有高效的优势 DNA复制系统的可用将使我们能够开始解决复制停滞之间的耦合问题 以及下游反应途径。最后，根据 MIRA 原则，我们将追求其他 DNA 代谢中令人着迷的问题可能而且毫无疑问会在我们的研究过程中出现。

DESCRIPTION: This proposal is centered on studies of the mechanism of DNA replication in eukaryotic cells (1) and on the consequences of replication dysfunction with regard to spontaneous and damage- induced mutagenesis (2), and to cell cycle checkpoint activation (3). Our primary approaches combine biochemical and biophysical analysis with genetic analysis in the yeast Saccharomyces cerevisiae, to gain insight in each of these three broadly defined pathways and their interconnectivity. Our studies are augmented with structural studies of the complex machineries that function in these processes. Proposed DNA replication studies are based on a strong record of progress in defining mechanisms of lagging strand DNA replication and Okazaki fragment maturation. Since Okazaki fragments represent by far the most frequent DNA discontinuities in all cells, it is imperative to understand the different layers of regulation of this process. Okazaki fragment maturation has primarily been studied in well-defined biochemical systems, in isolation from other events that occur at the replication fork. We propose to expand our studies within the context of a complete replication fork, which has been assembled at a yeast replication origin. Our mutagenesis studies will center on the main actors, DNA polymerase z and Rev1. On the one hand, Rev1 promotes DNA lesion bypass by Pol z; on the other hand, it limits the extent of mutagenesis by inhibiting Pol z-dependent DNA synthesis outside the narrow environment of the lesion. We will unravel the mechanism that underlies this dual regulatory function of Rev1. The primary focus of our checkpoint studies is on the two sensor protein kinases Mec1 and Tel1, the orthologs of human ATR and ATM, respectively. Biochemical and genetic studies will be combined with cryo-EM studies to understand how the basal activities of these unique protein kinases are activated. Furthermore, the advantage of having an efficient DNA replication system available will allow us to begin addressing the coupling between replication arrest and the downstream response pathways. Finally, in keeping with the MIRA principle, we will pursue other fascinating questions in DNA metabolism that may, and undoubtedly will arise during our investigations.