Chlorella Virus DNA Ligase: Structure and Mechanism

小球藻病毒 DNA 连接酶：结构和机制

• 批准号: 6526107
• 负责人: Stewart H Shuman
• 金额: $ 24.45万
• 依托单位: SLOAN-KETTERING INST CAN RESEARCH
• 依托单位国家: 美国
• 财政年份: 2001
• 资助国家: 美国
• 起止时间: 2001-08-01 至 2005-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6526107
• 关键词: adenosine triphosphate; crosslink; enzyme complex; enzyme mechanism; enzyme structure; genetic mapping; phosphoester ligase; protein structure function; structural biology

DESCRIPTION (provided by applicant): DNA ligases are ubiquitous enzymes that catalyze the essential final step in DNA replication and repair - the conversion of DNA nicks into phosphodiester bonds. The joining of a 5' phosphate strand to a 3' hydroxyl strand at the nick entails three chemical steps: (i) ligase reacts with ATP or NAD to form a covalent intermediate (ligase-adenylate) in which AMP is linked to a lysine on the enzyme; (ii) the AMP is transferred from the ligase to the 5' phosphate end to form a DNA-adenylate intermediate; (iii) ligase catalyzes attack by the 3'OH of the nick on DNA-adenylate to join the two polynucleotides and liberate AMP. Our goal is to understand how ligase reaction chemistry is catalyzed and how ligase recognizes "damaged" (i.e., nicked) DNA - using a eukaryotic virus DNA ligase as a model. Chlorella virus PBCV1 ligase is the smallest eukaryotic ATP-dependent ligase known (298-aa). It consists only of the catalytic core, unembellished by the large flanking domains that decorate cellular ligases. Nonetheless, Chlorella virus ligase sustains mitotic growth and DNA repair in yeast when it is the only ligase in the cell. As the minimal eukaryotic ligase, and one with an intrinsic nick-sensing function, the Chlorella virus enzyme presents an attractive target for structural and functional analysis. We have crystallized Chlorella virus DNA ligase and determined the structure of the covalent ligase-AMP reaction intermediate at 2.0 A resolution. Models of nick recognition and catalysis suggested by the ligase-AMP structure will be tested and clarified by the experiments outlined in this proposal. Our specific aims are: (1) to identify by structure-based mutagenesis the important functional groups of DNA ligase; (2) to define the interface between ligase-adenylate and nicked DNA using footprinting and crosslinking methods; (3) to determine by crystallography the structure of ligase-adenylate bound at a DNA nick; and (4) to determine the "ground-state" structure of ligase bound to ATP. The proposed experiments blend biochemistry, molecular genetics, and structural biology to elucidate how the chemical and conformational steps of the ligation pathway are coordinated. The findings will provide new insights into DNA damage recognition - an issue relevant to human health in light of the emerging genetic connections between DNA repair pathways and human cancer predisposition.

描述（由申请人提供）：DNA 连接酶是普遍存在的酶， 催化DNA复制和修复的最后一步—— DNA 切口转化为磷酸二酯键。 5' 的加入 磷酸链到切口处的 3' 羟基链需要三个化学物质 步骤： (i) 连接酶与 ATP 或 NAD 反应形成共价中间体 （连接酶-腺苷酸），其中 AMP 连接至酶上的赖氨酸； (二) AMP 从连接酶转移到 5' 磷酸末端，形成 DNA-腺苷酸中间体； (iii) 连接酶催化 3'OH 的攻击 在 DNA 腺苷酸上产生切口以连接两个多核苷酸并释放 AMP。 我们的目标是了解连接酶化学反应是如何催化的以及如何进行 连接酶使用真核病毒 DNA 识别“受损”（即带切口）DNA 连接酶作为模型。小球藻病毒PBCV1连接酶是最小的真核生物 已知 ATP 依赖性连接酶 (298-aa)。它仅由催化核心组成， 没有被装饰细胞连接酶的大侧翼结构域修饰。 尽管如此，小球藻病毒连接酶维持有丝分裂生长和 DNA 修复 当酵母是细胞中唯一的连接酶时。作为最小的真核连接酶， 以及一种具有内在切口感应功能的小球藻病毒酶 为结构和功能分析提供了一个有吸引力的目标。我们有 结晶小球藻病毒 DNA 连接酶并确定其结构 共价连接酶-AMP 反应中间体，分辨率为 2.0 A。 连接酶-AMP结构提出的切口识别和催化模型 将通过本提案中概述的实验进行测试和澄清。我们的 具体目标是：（1）通过基于结构的诱变来识别重要的 DNA连接酶的功能基团； (2) 定义之间的接口 使用足迹法和交联方法连接酶腺苷酸和切口 DNA； (3)通过晶体学测定结合酶-腺苷酸的结构 DNA 缺口； (4) 确定连接酶结合的“基态”结构 至 ATP。拟议的实验融合了生物化学、分子遗传学和 结构生物学阐明化学和构象步骤如何 连接途径是协调的。研究结果将提供新的见解 DNA损伤识别——一个与人类健康相关的问题 DNA修复途径与人类癌症之间新出现的遗传联系 倾向。