Dot1 and Histone H3 K79 Methylation in Nucleotide Excision Repair

智力优势：在真核细胞中，DNA被包装成染色质。染色质的基本结构是核小体，它由146个碱基对的DNA组成，包裹在一个蛋白质八聚体周围，蛋白质八聚体含有四个核心组蛋白中的每一个的两个分子，即H_2A、H_2B、H_3和H_4。正常的代谢活动和环境因素，如阳光和工业污染物，都会对细胞DNA造成损害。为了应对不断发生的DNA损伤，细胞配备了多条高度保守的DNA修复途径。一种重要的DNA修复途径，全球基因组修复(GGR)，负责修复整个基因组中巨大的DNA损伤。最近发现组蛋白H3赖氨酸79(H3K79)被组蛋白甲基转移酶Dot1甲基化是GGR所必需的。本项目的目标是通过使用真核模式生物啤酒酵母来研究Dot1对H3K79的甲基化是如何介导GGR的。在这个项目中将解决三个问题。首先，组蛋白H2B赖氨酸123的单一泛素化和组蛋白H4 N端尾部的一个碱性补丁已被证明反式激活Dot1。这些反式激活元件在GGR中的作用以及它们在调节GGR时如何与Dot1串扰将被确定。其次，H3K79甲基化所传递的GGR信号的性质将通过改变细胞中H3K79甲基化的不同状态来确定。还将确定GGR中核小体表面H3K79周围的氨基酸的作用。第三，甲基化H3K79招募的GGR因子将通过一种新的交联技术进行研究，该技术允许检测稀有蛋白质和丰富蛋白质之间的直接相互作用，即使这种相互作用在活细胞中可能是暂时的。H3K79招募的新的GGR特定因子将通过利用这种新的交联技术与质谱学相结合来确定。广泛影响：该项目包括一个重要的教育组成部分，大部分资金用于未来专业人员的研究培训，包括资助研究生和本科生在科学会议上展示他们的数据并在科学期刊上发表结果。该项目开发的一些新方法和产生的结果将在课堂上展示。独特的酵母突变体和开发的方法将与科学界共享。鉴于Dot1、H3K79甲基化、核小体结构和GGR过程在真核生物中高度保守，酵母研究的结果很可能适用于其他真核生物，从而增加对DNA修复过程和未修复损伤的后果的了解。

Intellectual merit:In eukaryotic cells, DNA is packaged into chromatin. The basic structure of chromatin is the nucleosome, which is comprised of 146 base pairs of DNA wrapped around a protein octamer containing two molecules of each of the four core histones, H2A, H2B, H3, and H4. Both normal metabolic activities and environmental factors such as sunlight and industrial pollutants can cause damage to cellular DNA. To contend with the constantly occurring DNA damage, cells are equipped with multiple, highly conserved DNA repair pathways. One important DNA repair pathway, global genomic repair (GGR), is responsible for repairing bulky DNA lesions throughout the genome. It was discovered very recently that methylation of histone H3 lysine 79 (H3K79) by Dot1, a histone methyltransferase, is required for GGR. The goal of this project is to investigate how methylation of H3K79 by Dot1 mediates GGR by using the eukaryotic model organism brewer's yeast, Sacharomyces cerevisiae. Three questions will be addressed in this project. First, mono-ubiquitination of histone H2B lysine 123 and a basic patch in the N-terminal tail of histone H4 have been shown to trans-activate Dot1. The roles of these trans-activating elements in GGR and how they crosstalk with Dot1 in modulating GGR will be determined. Second, the nature of the GGR signal conferred by H3K79 methylation will be determined by changing the different states of H3K79 methylation in the cell. The roles of amino acids surrounding H3K79 on the nucleosome surface in GGR will also be determined. Third, GGR factors recruited by methylated H3K79 will be investigated by using a novel crosslinking technique that allows for detection of direct interaction between a rare protein and an abundant protein even if the interaction may be transient in living cells. New GGR-specific factors recruited by H3K79 will be identified by utilizing this novel crosslinking technique in combination with mass spectrometry.Broader impacts:This project includes a significant educational component, with most of the funding used for research training of future professionals, including funding for graduate and undergraduate students to present their data at scientific meetings and publish results in scientific journals. Some of the new methods developed and findings generated from the project will be presented in the classroom. Unique yeast mutants and methodologies developed will be shared with the scientific community. Given the fact that Dot1, H3K79 methylation, nucleosome structure and the GGR process are highly conserved among eukaryotes, the findings generated from the yeast studies are likely to be applicable to other eukaryotic species, thus increasing the understanding of DNA repair processes, and the consequences of unrepaired damage.