Mechanisms of DNA replication and maintenance in eukaryotes

真核生物中 DNA 复制和维持的机制

• 批准号: 10625860
• 负责人: PETER M BURGERS
• 金额: $ 69.74万
• 依托单位: WASHINGTON UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2016
• 资助国家: 美国
• 起止时间: 2016-06-01 至 2026-05-31
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10625860
• 关键词: ATR gene; Address; Biochemical; Bypass; Cell Cycle Checkpoint; Cells; Complex; Coupling; Cryoelectron Microscopy; DNA; DNA Damage; DNA Maintenance; DNA biosynthesis; DNA lesion; DNA metabolism; DNA replication fork; DNA-Directed DNA Polymerase; Defect; Environment; Eukaryota; Eukaryotic Cell; Event; Functional disorder; Genetic study; Human; Investigation; Lesion; Malignant Neoplasms; Mutagenesis; Okazaki fragments; Orthologous Gene; Pathway interactions; Patients; Process; Protein Kinase; Regulation; Replication Origin; Risk; Saccharomyces cerevisiae; System; TEL1 Gene; Yeasts; biophysical analysis; fascinate; genetic analysis; genetic information; insight; model organism; response; sensor

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.

内容描述：本项目主要研究真核生物中DNA复制的机制， 细胞（1）和复制功能障碍的后果方面的自发和损害- 诱导诱变（2），和细胞周期检查点激活（3）。我们的主要方法结合了联合收割机 生物化学和生物物理分析与酵母酿酒酵母中的遗传分析，以获得 深入了解这三种广泛定义的途径及其相互联系。我们的研究是 通过对在这些过程中发挥作用的复杂机制的结构研究来增强。提出 DNA复制的研究是基于一个强有力的记录，在确定机制的滞后链 DNA复制和冈崎片段成熟。因为冈崎的碎片代表了迄今为止 在所有细胞中频繁的DNA不连续性，必须了解不同层次的调控， 这个过程冈崎片段的成熟主要是在明确定义的生化系统中研究的， 与复制分叉处发生的其他事件隔离。我们建议扩大我们的研究范围， 在一个实施例中，复制叉是一个完整的复制叉，它已经在酵母复制起点组装。我们 诱变研究将集中在主要的参与者，DNA聚合酶Z和Rev 1。一方面，Rev 1 促进DNA损伤绕过Pol z;另一方面，它通过抑制Pol z限制诱变的程度。 在病变的狭窄环境之外的z依赖性DNA合成。我们会解开 这是Rev 1的双重调节功能的基础。我们检查点研究的主要重点是 两种传感蛋白激酶Mec 1和Tel 1，分别是人ATR和ATM的直系同源物。 生物化学和遗传学研究将与冷冻电镜研究相结合，以了解基础的 这些独特的蛋白激酶的活性被激活。此外，具有高效的 可用的DNA复制系统将使我们开始解决复制停滞之间的耦合 以及下游的反应途径。最后，根据MIRA原则，我们将寻求其他 在我们的研究中，DNA代谢中的一些有趣的问题可能会出现，毫无疑问也会出现。

项目成果

The fidelity of DNA replication, particularly on GC-rich templates, is reduced by defects of the Fe-S cluster in DNA polymerase δ.

• DOI: 10.1093/nar/gkab371
• 发表时间: 2021-06-04
• 期刊: Nucleic acids research
• 影响因子: 14.9
• 作者: Kiktev DA;Dominska M;Zhang T;Dahl J;Stepchenkova EI;Mieczkowski P;Burgers PM;Lujan S;Burkholder A;Kunkel TA;Petes TD
• 通讯作者: Petes TD

Yeast DNA polymerase ζ maintains consistent activity and mutagenicity across a wide range of physiological dNTP concentrations.

• DOI: 10.1093/nar/gkw1149
• 发表时间: 2017-02-17
• 期刊: Nucleic acids research
• 影响因子: 14.9
• 作者: Kochenova OV;Bezalel-Buch R;Tran P;Makarova AV;Chabes A;Burgers PM;Shcherbakova PV
• 通讯作者: Shcherbakova PV

Arranging eukaryotic nuclear DNA polymerases for replication: Specific interactions with accessory proteins arrange Pols α, δ, and ϵ in the replisome for leading-strand and lagging-strand DNA replication.

• DOI: 10.1002/bies.201700070
• 发表时间: 2017-08
• 期刊: BioEssays : news and reviews in molecular, cellular and developmental biology
• 作者: Kunkel TA;Burgers PMJ
• 通讯作者: Burgers PMJ

Eukaryotic DNA Replication Fork.

• DOI: 10.1146/annurev-biochem-061516-044709
• 发表时间: 2017-06-20
• 期刊: Annual review of biochemistry
• 影响因子: 16.6
• 作者: Burgers PMJ;Kunkel TA
• 通讯作者: Kunkel TA

The Dimeric Architecture of Checkpoint Kinases Mec1ATR and Tel1ATM Reveal a Common Structural Organization.

• DOI: 10.1074/jbc.m115.708263
• 发表时间: 2016-06-24
• 期刊: The Journal of biological chemistry
• 作者: Sawicka M;Wanrooij PH;Darbari VC;Tannous E;Hailemariam S;Bose D;Makarova AV;Burgers PM;Zhang X
• 通讯作者: Zhang X