Mechanisms of DNA replication and maintenance in eukaryotes

描述：这项建议主要针对真核生物中DNA复制机制的研究。 细胞(1)和复制功能障碍对自发性和损伤的后果- 诱变(2)，细胞周期检查点激活(3)。我们的主要方法结合了 酿酒酵母的生化、生物物理分析和遗传分析，以获得 对这三条广泛定义的路径及其相互联系的洞察。我们的研究是 增加了对在这些过程中起作用的复杂机械的结构研究。建议 DNA复制研究是基于在确定滞后链机制方面取得进展的有力记录 DNA复制和Okazaki片段成熟。因为到目前为止冈崎的碎片代表了 在所有细胞中频繁的DNA中断，了解不同层次的调控是当务之急 这一过程。冈崎片段的成熟主要是在定义良好的生化系统中进行的， 与发生在复制分叉处的其他事件隔离。我们建议将我们的研究扩展到 已在酵母复制起始处组装的完整复制叉子的上下文。我们的 突变研究将集中在主要因素DNA聚合酶z和Rev1上。一方面，Rev1 通过Pol z促进DNA损伤旁路；另一方面，它通过抑制Pol来限制突变的程度 在狭窄的病变环境之外，Z依赖的DNA合成。我们将解开这一机制 这就是Rev1的双重调控功能的基础。我们检查站研究的主要焦点是 两个传感器蛋白激酶Mec1和Tel1，分别是人类ATR和ATM的同源基因。 生化和遗传学研究将与冷冻-EM研究相结合，以了解基础 这些独特的蛋白激酶的活性被激活。此外，拥有高效的 可用的DNA复制系统将使我们能够开始解决复制停滞之间的耦合 以及下游反应通路。最后，为了与米拉原则保持一致，我们将寻求其他 DNA新陈代谢方面的有趣问题，在我们的研究过程中可能会出现，而且毫无疑问会出现。

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.