A New Structural Architecture for DNA Processing

智力上的优点。DNA不断受到细胞和环境DNA破坏剂的化学攻击。为了保持基因组的完整性，所有生物体都拥有损伤特异性DNA修复蛋白，面临着在大量“正常”DNA中定位特定病变的共同挑战。这项研究通过对新发现的蜡状芽孢杆菌中一种名为AlkD的酶的结构-功能分析来解决这个基本问题；这种酶属于一组被称为DNA糖基酶的蛋白质，它将识别某些经过化学修饰的DNA碱基。DNA糖基酶通过检测和去除DNA中的修饰碱基来启动特定的修复途径。糖基酶专一性的基础尚不清楚，但被认为部分原因是受损碱基本身的不稳定性。在PI的实验室中测定的AlkD与受损DNA结合的X射线晶体结构表明，AlkD利用一种新的DNA结合结构，称为热重复，以一种前所未有的机制捕获DNA损伤。最近发现热重复蛋白在几种DNA损伤反应蛋白中形成扩展结构域。因此，AlkD代表了一种新的DNA加工酶，它提供了一种机会来确定1)热重复进行DNA损伤的分子细节，以及2)损伤碱基识别和移除的化学和物理决定因素。结合结构生物学方法，包括X射线结晶学和核磁共振，将获得与DNA损伤的碱基D络合物的高分辨结构，结构数据将指导DNA结合和碱基切除活性的生化分析。这项研究计划的结果将被放在PI的实验室和其他地方正在进行的DNA修复研究的背景下。学生通常只在课堂上接触到X射线结晶学的理论方面和先进的结构技术。该项目将通过在实验室和课堂上为所有级别的学生提供实用的结构生物学培训，将研究和教育结合起来。PI的蛋白质化学课程将包含一个动手操作的X射线结晶学模块，为本科生和研究生提供直接参与蛋白质结构测定的所有方面的独特机会。作为AlkD研究计划的一部分，学生将帮助蛋白质-DNA复合体的结晶、结构解决、模型建立、精炼和验证，并将在PI的实验室中由PI和研究生分组指导。与该奖项相关的研究生将指导本科生在PI的实验室完成他们自己的独立项目，以完成研究课程的学分。因此，除了本科生获得X射线结晶学的实际研究经验外，研究生还将通过在实验室指导学生获得宝贵的教学经验。PI与文理学院和范德比尔特大学医学院的五个研究中心建立了联系，为本科生、研究生、博士后研究员和访问科学家提供了丰富的研究和教学环境。

Intellectual merit. DNA is under constant chemical assault from cellular and environmental DNA damaging agents. To maintain integrity of their genomes, all organisms possess damage-specific DNA repair proteins faced with the common challenge of locating a particular lesion within a vast excess of "normal" DNA. This research addresses this fundamental question through structure-function analysis of a newly discovered enzyme from Bacillus cereus named AlkD; this enzyme, which belongs to a group of proteins referred-to as DNA glycosylases, will recognize certain DNA bases that have been chemically modified. DNA glycosylases initiate a particular repair pathway by detecting and removing the modified bases from DNA. The basis for glycosylase specificity is poorly understood, but is believed to result in part from instability of the damaged bases themselves. X-ray crystal structures of AlkD bound to damaged DNA, determined in the PI's laboratory, revealed that AlkD utilizes a new DNA binding architecture, referred-to as a HEAT repeat, to capture DNA damage by an unprecedented mechanism. HEAT repeat proteins have recently been identified to form expansive domains within several DNA damage response proteins. Thus, AlkD represents a new class of DNA processing enzyme that offers an opportunity to determine 1) the molecular details by which HEAT repeats engage DNA damage, and 2) the chemical and physical determinants of damaged base recognition and removal. A combination of structural biology methods, including X-ray crystallography and NMR, will be used to obtain high-resolution structures of AlkD complexes with damaged DNA, and the structural data will guide biochemical analyses of DNA binding and base excision activities. Results from this research program will be placed in the context of on-going DNA repair research in the PI's laboratory and elsewhere.Broader impact. Students are typically only exposed to the theoretical aspects of X-ray crystallography and advanced structural techniques in a lecture setting. This project will integrate research and education by providing practical structural biology training to students at all levels in both the laboratory and the classroom. A hands-on X-ray crystallography module will be incorporated into the PI's protein chemistry course to provide undergraduate and graduate students with the unique opportunity to participate directly in all aspects of protein structure determination. Students will aid in crystallization, structure solution, model building, refinement, and validation of protein-DNA complexes as part of the AlkD research program, and will be directed in groups by both the PI and graduate students in the PI's laboratory. Graduate students associated with this award will direct undergraduates working on their own independent projects in the PI's laboratory in fulfillment of research course credit. Thus, in addition to undergraduates gaining practical research experience in X-ray crystallography, graduate students will gain invaluable teaching experience by guiding students in the laboratory. The PI's affiliations with the College of Arts and Science and five research centers in the medical school at Vanderbilt University offer a rich research and teaching environment involving undergraduates, graduate students postdoctoral fellows, and visiting scientists.