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EPIGENETIC CONTROL OF NHEJ DNA REPAIR

NHEJ DNA 修复的表观遗传控制

基本信息

批准号：
8634733
负责人：
Robert A Hromas
金额：
$ 29.43万
依托单位：
UNIVERSITY OF FLORIDA
依托单位国家：
美国
项目类别：
财政年份：
2010
资助国家：
美国
起止时间：
2010-01-01 至 2016-03-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/8634733
关键词：
Adriamycin PFS Affect Biological Assay Breast Cancer Cell CHES1 gene Cancer Etiology Cancer cell line Chromatin Chromosomal Breaks Chromosomal translocation Chromosomes Clinical Code Complex DNA DNA Double Strand Break DNA Repair DNA Repair Pathway DNA ligase IV Data Double Strand Break Repair Epigenetic Process Event Genomic Instability Histones Human Incidence Ionizing radiation Link Lysine Malignant Neoplasms Mediating Methylation Methyltransferase Molecular Movement Mutate Mutation NBS1 gene Nonhomologous DNA End Joining Organism Pathway interactions Pharmaceutical Preparations Phosphorylation Play Poison Process Proteins Radiation therapy Recruitment Activity Regulation Resistance Role SET Domain Series Site Structure Structure-Activity Relationship Superhelical DNA Testing Time Topoisomerase II Translating Transposase Yeasts base cancer cell chromatin immunoprecipitation chromatin modification clinically relevant drug testing endonuclease histone methyltransferase histone modification improved in vitro Assay inhibitor/antagonist insight mutant novel nuclease prevent public health relevance radiation resistance repaired screening virtual

项目摘要

DESCRIPTION (provided by applicant): Transposase activity was thought to be extinct in humans because DNA movement can be deleterious in higher organisms, resulting in genomic instability and perhaps malignancy. However, we isolated a human transposase protein termed Metnase that had both a Transposase domain that functions as an endonuclease and a SET histone methylase domain. The Transposase domain has preferential endonuclease activity for supercoiled DNA, and the SET domain was able to methylate histone 3 at lysine 36, associated with open chromatin. Metnase enhances resistance to radiation therapy, and improves repair of DNA double strand breaks (DSBs) via the non-homologous end-joining pathway (NHEJ). Both the SET and Transposase domains were required for the NHEJ repair activity. Metnase was found to interact with the NBS1, an early NHEJ repair pathway component, DNA Ligase IV, a final component of the NHEJ pathway, and Pso4, an uncharacterized DSB repair component. We found that Metnase decreases the incidence of long deletions at the repaired DSB junction site. Metnase is phosphorylated at S495, and this is essential for its NHEJ repair activity. We show that Metnase decreases the rate of inter-chromosomal translocation when there are simultaneous DSBs on distinct chromosomes, consistent with its NHEJ repair activity. We also found that Metnase also mediates resistance to Topo II poisons, which cause DSBs, in cancer cell lines. Thus, Metnase is a novel component of the NHEJ repair pathway, and links histone modification to DSB repair. The mechanism by which Metnase improves DNA repair is unknown, but its SET histone methylase domain is essential to its NHEJ activity. Recent studies in yeast indicate that the modification of chromatin, including histone methylation, may be an important part of DSB repair. However, despite its potential importance, the connection between such epigenetic chromatin modifications and NHEJ DSB repair is not well defined. Using a novel ChIP assay to analyze proteins associated with a single defined DSB, we found that Metnase localized to that DSB, and dimethylated H3K36 there. We also found evidence that dimethylated H3K36 may recruit early NHEJ components, such as ATM and the MRN complex, to the DSB. Based on these data, we hypothesize that Metnase plays an important role in the epigenetic regulation of NHEJ DSB repair. This hypothesis will be explored in four aims that translate molecular mechanisms to clinical relevance- 1) What are the structures of Metnase that are essential for its histone methylase activity? 2) What are the histone alterations Metnase makes around a DSB sites? 3) What is the mechanism by which Metnase's histone methylation enhances DSB repair? 4) Can the histone methylase activity of Metnase be exploited clinically?

描述（由申请人提供）：人们认为转座酶活性在人类中已经灭绝，因为 DNA 运动对高等生物体可能是有害的，导致基因组不稳定，甚至可能导致恶性肿瘤。然而，我们分离出了一种名为 Metnase 的人类转座酶蛋白，它具有充当核酸内切酶功能的转座酶结构域和 SET 组蛋白甲基化酶结构域。转座酶结构域对超螺旋 DNA 具有优先的核酸内切酶活性，而 SET 结构域能够在赖氨酸 36 处甲基化与开放染色质相关的组蛋白 3。 Metnase 增强对放射治疗的抵抗力，并通过非同源末端连接途径 (NHEJ) 改善 DNA 双链断裂 (DSB) 的修复。 NHEJ 修复活动需要 SET 和转座酶结构域。 Metnase 被发现与 NBS1（一种早期 NHEJ 修复途径成分）、DNA 连接酶 IV（NHEJ 途径的最终成分）和 Pso4（一种未表征的 DSB 修复成分）相互作用。我们发现 Metnase 降低了修复的 DSB 连接位点长缺失的发生率。 Metnase 在 S495 处被磷酸化，这对于其 NHEJ 修复活性至关重要。我们发现，当不同染色体上同时存在 DSB 时，Metnase 会降低染色体间易位的速率，这与其 NHEJ 修复活性一致。我们还发现 Metnase 还能介导癌细胞系中对 Topo II 毒物的抵抗力，这种毒物会导致 DSB。因此，Metnase 是 NHEJ 修复途径的一个新成分，它将组蛋白修饰与 DSB 修复联系起来。 Metnase 改善 DNA 修复的机制尚不清楚，但其 SET 组蛋白甲基化酶结构域对其 NHEJ 活性至关重要。最近在酵母中的研究表明，染色质的修饰，包括组蛋白甲基化，可能是 DSB 修复的重要组成部分。然而，尽管这种表观遗传染色质修饰和 NHEJ DSB 修复之间的联系具有潜在的重要性，但尚不清楚。使用新型 ChIP 测定来分析与单个定义的 DSB 相关的蛋白质，我们发现 Metnase 定位于该 DSB，并在那里二甲基化 H3K36。我们还发现证据表明二甲基化 H3K36 可能会将早期 NHEJ 成分（例如 ATM 和 MRN 复合物）招募到 DSB。基于这些数据，我们假设 Metnase 在 NHEJ DSB 修复的表观遗传调控中发挥重要作用。该假设将在四个目标中进行探索，将分子机制转化为临床相关性 - 1) 对于其组蛋白甲基化酶活性至关重要的 Metnase 结构是什么？ 2) Metnase 在 DSB 位点周围进行哪些组蛋白改变？ 3）Metnase的组蛋白甲基化增强DSB修复的机制是什么？ 4）Metnase的组蛋白甲基化酶活性可以应用于临床吗？

项目成果

期刊论文数量（4）

专著数量（0）

科研奖励数量（0）

会议论文数量（0）

专利数量（0）

Overview for the histone codes for DNA repair.

DOI：
10.1016/b978-0-12-387665-2.00008-0
发表时间：
2012
期刊：
PROGRESS IN MOLECULAR BIOLOGY AND TRANSLATIONAL SCIENCE
影响因子：
0
作者：
Williamson, Elizabeth A.;Wray, Justin W.;Bansal, Pranshu;Hromas, Robert
通讯作者：
Hromas, Robert

Adenomatous polyposis coli-mediated accumulation of abasic DNA lesions lead to cigarette smoke condensate-induced neoplastic transformation of normal breast epithelial cells.

腺瘤性息肉病大肠杆菌介导的无碱基 DNA 损伤的积累导致香烟烟雾冷凝物诱导正常乳腺上皮细胞的肿瘤性转化。

DOI：
10.1593/neo.13176
发表时间：
2013
期刊：
Neoplasia (New York, N.Y.)
影响因子：
0
作者：
Jaiswal,ArunaS;Panda,Harekrushna;Pampo,ChristineA;Siemann,DietmarW;Gairola,CGary;Hromas,Robert;Narayan,Satya
通讯作者：
Narayan,Satya

Cellular Responses to Widespread DNA Replication Stress.