A New Structural Architecture for DNA Processing

DNA 处理的新结构架构

• 批准号: 1122098
• 负责人: Brandt Eichman
• 金额: $ 69万
• 依托单位: Vanderbilt University
• 依托单位国家: 美国
• 项目类别: Standard Grant
• 财政年份: 2011
• 资助国家: 美国
• 起止时间: 2011-08-15 至 2015-07-31
• 项目状态: 已结题
• 来源: https://www.nsf.gov/awardsearch/showAward?AWD_ID=1122098&HistoricalAwards=false
• 关键词: New; Structural; Architecture; DNA; Processing

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.

智力优点。 DNA受到细胞和环境DNA损伤剂的恒定化学攻击。为了维持其基因组的完整性，所有生物体都具有损伤特异性DNA修复蛋白面临的损害特异性DNA修复蛋白，该蛋白面临着将特定病变定位在巨大的“正常” DNA中的常见挑战。这项研究通过对熟食芽孢杆菌的新发现的酶的结构 - 功能分析来解决这个基本问题。该酶属于称为DNA糖基酶的一组蛋白质，将识别某些已化学修饰的DNA碱基。 DNA糖基酶通过检测并从DNA中去除修饰的碱基来启动特定的修复途径。糖基酶特异性的基础知之甚少，但被认为部分是由于受损碱基本身的不稳定。 ALKD的X射线晶体结构与受损的DNA结合，在PI的实验室中确定，ALKD利用一种新的DNA结合结构，称为热重复，通过前所未有的机制捕获DNA损伤。最近已经确定了热重复蛋白在几种DNA损伤反应蛋白中形成膨胀域。因此，ALKD代表了一种新的DNA加工酶，该酶提供了确定热量重复引起DNA损伤的分子细节的机会，以及2）2）基础识别和去除的化学和物理决定因素。结构生物学方法（包括X射线晶体学和NMR）的结合将用于获得具有损坏DNA的ALKD复合物的高分辨率结构，结构数据将指导DNA结合和碱基切除活性的生化分析。该研究计划的结果将放置在PI实验室和其他地方正在进行的DNA维修研究的背景下。通常，学生仅在讲座环境中暴露于X射线晶体学和高级结构技术的理论方面。该项目将通过向实验室和课堂的各个级别的学生提供实用的结构生物学培训来整合研究和教育。动手X射线晶体学模块将被纳入PI的蛋白质化学课程中，以向本科生和研究生提供直接参与蛋白质结构确定各个方面的独特机会。作为ALKD研究计划的一部分，学生将帮助结晶，结构解决方案，模型建设，改进和验证蛋白质-DNA复合物，并将由PI实验室的PI和研究生组成。与该奖项相关的研究生将指导本科生在PI的实验室中从事自己的独立项目，以实现研究课程学分。因此，除了获得X射线晶体学上实践研究经验的大学生外，研究生还将通过指导实验室的学生获得宝贵的教学经验。 PI与艺术与科学学院以及范德比尔特大学医学院的五个研究中心的隶属关系提供了丰富的研究和教学环境，包括大学生，研究生博士后研究员以及来访的科学家。