A New Structural Architecture for Recognition of DNA Damage

一种识别 DNA 损伤的新结构体系

• 批准号: 1517695
• 负责人: Brandt Eichman
• 金额: $ 66万
• 依托单位: Vanderbilt University
• 依托单位国家: 美国
• 项目类别: Continuing Grant
• 财政年份: 2015
• 资助国家: 美国
• 起止时间: 2015-08-01 至 2019-07-31
• 项目状态: 已结题
• 来源: https://www.nsf.gov/awardsearch/showAward?AWD_ID=1517695&HistoricalAwards=false
• 关键词: New; Structural; Architecture; Recognition; DNA

This project is funded jointly by the Genetic Mechanisms Cluster in the Division of Molecular and Cellular Biosciences in the Directorate for Biological Sciences and the Chemistry of Life Processes Program in the Division of Chemistry in the Directorate of Mathematical and Physical Sciences.Chemical modification and damage of DNA from a continual onslaught of cellular and environmental agents alters genetic information and threatens all aspects of cellular function. DNA repair proteins exist in all organisms to remove damaged DNA and protect the integrity of the genome. This research aims to understand at the atomic level how DNA repair proteins locate a particular type of DNA damage and initiate the process of fixing it. This program will enhance the educational benefits to society through 1) integration of research and education in the laboratory and the classroom, 2) vertical integration at all levels of training--from undergraduates to postdoctoral associates, 3) practical structural biology experience for undergraduates, 4) community outreach, and 5) participation by women and underrepresented groups, and 6) integration of science and art to enhance scientific communication. The close proximity of the College of Arts and Science and the School of Medicine at Vanderbilt University provides an exceptional collaborative training environment. Practical hands-on X-ray crystallography laboratory modules have been incorporated into the PI's courses to provide students with the unique opportunity to directly participate in all aspects of protein structure determination. All personnel associated with this research are involved in recruiting students from underrepresented groups from regional institutions, and the PI is involved in outreach through interactions with local high, middle, and elementary school students.The long term goal of this research is to determine the mechanisms by which DNA repair enzymes locate and repair aberrant DNA. This research focuses on the structures and functions of a relatively new superfamily of DNA glycosylases, represented by the Bacillus cereus AlkD enzyme, that catalyzes the excision of cationic alkylated DNA nucleobases, including N3-methyladenine (3mA) and N7-methylguanine (7mG), which are among the most prevalent forms of DNA damage. Because of their inherent instability, the basis for recognition and removal of cationic alkylbases from DNA is unknown. The AlkD-related enzymes are unique in that they are the only DNA glycosylases specific for cationic lesions and with the ability to excise bulky modifications. Additionally, these enzymes are constructed from a tandem helical repeat architecture that has emerged as an important nucleic acid processing platform in chromatin remodeling and DNA damage response proteins. The PI's group has established that unlike other DNA glycosylases, AlkD does not need to flip the nucleobase target out of the DNA duplex prior to catalysis. Thus, the AlkD superfamily is an ideal system to study the fundamental requirements for base excision repair of alkylation damage repair, with strong potential to reveal novel mechanistic insight into DNA damage recognition. Four specific aims will integrate structural and computational biology, biochemistry, and genetic approaches to 1) determine the physicochemical features that underlie recognition and excision of cationic N3- and N7-alkylpurines, 2) elucidate the diversity among the AlkC/D superfamily that defines substrate specificity, 3) investigate how this unique damage recognition platform can excise bulky DNA adducts, and 4) understand the apparent redundancy between alkylpurine DNA glycosylase activities in Bacillus and to determine if AlkD participates in alternative repair pathways.

该项目由生物学科学局和分子生物科学的划分中的遗传机制集群共同资助，生命过程的化学过程中的化学过程中的化学过程分工是数学和物理科学局，从而使DNA的化学修改和损害不断地损害细胞和环境环境的损害。所有生物体中都存在DNA修复蛋白，以去除受损的DNA并保护基因组的完整性。这项研究旨在了解原子水平的DNA修复蛋白如何找到特定类型的DNA损伤并启动修复它的过程。该计划将通过1）在实验室和课堂上的研究和教育的整合增强社会的教育益处，2）在各个培训的各个级别的垂直整合 - 从本科生到博士后伙伴，3）实用的结构生物学经验，用于培训，4）妇女社区外界的社区外界，以及5）妇女和不足的团体的参与，并促进了科学和6）的科学和6）。范德比尔特大学的艺术与科学学院和医学院的密切接近，提供了出色的协作培训环境。实用的动手X射线晶体学实验室模块已被整合到PI的课程中，为学生提供了直接参与蛋白质结构确定各个方面的独特机会。与这项研究相关的所有人员都参与了来自地区机构代表性不足的群体的招募学生，而PI通过与当地高层，中学和小学生的互动来参与外展活动。这项研究的长期目标是确定DNA修复酶定位酶和维修异常DNA的机制。这项研究侧重于以Cereus cereus Alkd酶为代表的相对较新的DNA糖基化酶的结构和功能，这些酶催化了阳离子烷基化DNA核苷酸酶的切除，包括N3-甲基丙氨酸（3MA）和N7-甲基鸟嘌呤（7mthely（7mger））（包括N3-甲基丙氨酸（3MA）（7mger）（7mgg），这是e;由于它们固有的不稳定性，从DNA中识别和去除阳离子烷基基的基础是未知的。与ALKD相关的酶的独特之处在于它们是阳离子病变特异性的唯一DNA糖基酶，并且具有切除笨重修饰的能力。此外，这些酶是由串联螺旋重复体系结构构建的，该酶已成为染色质重塑和DNA损伤反应蛋白中重要的核酸加工平台。 PI组已经确定，与其他DNA糖基酶不同，ALKD在催化前不需要将核碱酶靶标从DNA双链体中翻转出来。因此，ALKD超家族是研究基础切除烷基化损伤修复的基本要求的理想系统，具有强大的潜力，可以揭示对DNA损伤识别的新机械洞察力。 Four specific aims will integrate structural and computational biology, biochemistry, and genetic approaches to 1) determine the physicochemical features that underlie recognition and excision of cationic N3- and N7-alkylpurines, 2) elucidate the diversity among the AlkC/D superfamily that defines substrate specificity, 3) investigate how this unique damage recognition platform can excise bulky DNA adducts, and 4) understand the芽孢杆菌中烷基丁嘌呤DNA糖基化酶活性之间的明显冗余性，并确定ALKD是否参与替代修复途径。