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.

智力上的优点。 DNA 不断受到细胞和环境 DNA 损伤剂的化学攻击。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，它们面临着在大量过量的“正常”DNA 中定位特定损伤的共同挑战。本研究通过对蜡状芽孢杆菌中新发现的一种名为 AlkD 的酶进行结构功能分析来解决这一基本问题。这种酶属于一组称为 DNA 糖基化酶的蛋白质，可以识别某些经过化学修饰的 DNA 碱基。 DNA 糖基化酶通过检测并去除 DNA 中的修饰碱基来启动特定的修复途径。糖基化酶特异性的基础尚不清楚，但据信部分是由于受损碱基本身的不稳定性造成的。 PI 实验室测定的 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 HEAT 重复）以前所未有的机制捕获 DNA 损伤。最近已发现 HEAT 重复蛋白可在多种 DNA 损伤反应蛋白内形成广泛的结构域。因此，AlkD 代表了一类新型 DNA 加工酶，它提供了确定 1) HEAT 重复引起 DNA 损伤的分子细节，以及 2) 受损碱基识别和去除的化学和物理决定因素的机会。结合结构生物学方法，包括X射线晶体学和NMR，将用于获得AlkD与受损DNA复合物的高分辨率结构，并且结构数据将指导DNA结合和碱基切除活性的生化分析。该研究计划的结果将置于 PI 实验室和其他地方正在进行的 DNA 修复研究的背景下。产生更广泛的影响。学生通常仅在讲座中接触到 X 射线晶体学的理论知识和先进的结构技术。该项目将通过在实验室和课堂上为各级学生提供实用的结构生物学培训来整合研究和教育。 PI 的蛋白质化学课程中将纳入 X 射线晶体学实践模块，为本科生和研究生提供直接参与蛋白质结构测定各个方面的独特机会。作为 AlkD 研究计划的一部分，学生将协助蛋白质-DNA 复合物的结晶、结构解析、模型构建、完善和验证，并将由 PI 和 PI 实验室的研究生分组指导。与该奖项相关的研究生将指导本科生在 PI 实验室中开展自己的独立项目，以完成研究课程学分。因此，除了本科生获得 X 射线晶体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 与范德比尔特大学文理学院和医学院的五个研究中心的附属关系提供了丰富的研究和教学环境，涉及本科生、研究生、博士后研究员和访问科学家。