A New Structural Architecture for Recognition of DNA Damage

这个项目是由生物科学局分子和细胞生物科学部的遗传机制组和数学和物理科学局化学部的生命过程化学计划共同资助的。细胞和环境介质的持续攻击对DNA的化学修饰和破坏会改变遗传信息，并威胁到细胞功能的方方面面。DNA修复蛋白存在于所有生物体中，用于去除受损的DNA并保护基因组的完整性。这项研究的目的是在原子水平上了解DNA修复蛋白如何定位特定类型的DNA损伤并启动修复过程。该计划将通过1)实验室和课堂研究和教育的整合，2)各级培训的垂直整合--从本科生到博士后助理，3)为本科生提供实用的结构生物学体验，4)社区推广，以及5)妇女和代表不足的群体的参与，以及6)整合科学和艺术以加强科学交流，从而提高对社会的教育效益。范德比尔特大学文理学院和医学院的距离很近，提供了一个非常好的协作培训环境。在PI的课程中加入了实际操作的X射线结晶学实验室模块，为学生提供了直接参与蛋白质结构测定的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从地区机构中代表性不足的群体中招收学生，而PI则通过与当地高中、初中和小学生的互动参与了推广活动。这项研究的长期目标是确定DNA修复酶定位和修复异常DNA的机制。本研究的重点是以蜡状芽孢杆菌碱性D酶为代表的一个相对较新的DNA糖基酶超家族的结构和功能，它催化阳离子烷基化DNA碱基的切除，包括N3-甲基腺嘌呤(3 MA)和N7-甲基鸟嘌呤(7 Mg)，这是最常见的DNA损伤形式之一。由于其固有的不稳定性，识别和去除DNA中的阳离子烷基的基础尚不清楚。与碱D相关的酶是独特的，因为它们是唯一专用于阳离子损伤的DNA糖基酶，并具有切除大量修饰的能力。此外，这些酶是由串联螺旋重复结构构建的，该结构已成为染色质重塑和DNA损伤反应蛋白的重要核酸处理平台。PI的小组已经确定，与其他DNA糖基酶不同，AlkD不需要在催化之前将碱基靶标从DNA双链中翻转出来。因此，AlkD超家族是研究烷基化损伤修复中碱基切除修复的基本要求的理想系统，具有揭示DNA损伤识别的新机制的强大潜力。四个具体目标将结合结构和计算生物学、生物化学和遗传学方法，以1)确定识别和切除阳离子N3-和N7-烷基嘌呤的物理化学特征，2)阐明定义底物特异性的AlkC/D超家族之间的多样性，3)研究这个独特的损伤识别平台如何切除庞大的DNA加合物，以及4)了解芽孢杆菌中烷基嘌呤DNA糖基酶活性之间的明显冗余，并确定AlkD是否参与了替代的修复途径。

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.