Chlorella Virus DNA Ligase: Structure and Mechanism

描述（由申请人提供）：DNA连接酶是普遍存在的酶， 催化DNA复制和修复的关键最后一步- DNA缺口转化为磷酸二酯键。加入一个5' 在切口处将磷酸链连接到3'羟基链需要三种化学物质 步骤：（i）连接酶与ATP或NAD反应形成共价中间体 （ii）其中AMP与酶上的赖氨酸连接的连接酶-腺苷酸; AMP从连接酶转移到5'磷酸末端，形成一个连接酶。 DNA-腺苷酸中间体;（iii）连接酶催化由DNA-腺苷酸中间体的3 'OH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反应化学是如何催化的，以及如何 连接酶识别“受损的”（即，有切口的）DNA -使用真核病毒DNA 连接酶作为模型。小球藻病毒PBCV 1连接酶是真核生物中最小的 已知ATP依赖性连接酶（298-aa）。它只由催化核心组成， 未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， 酵母，当它是细胞中唯一的连接酶时。作为最小真核连接酶， 另一种是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有吸引力的目标。我们有 结晶小球藻病毒DNA连接酶，并确定结构的 共价连接酶-AMP反应中间体。 由连接酶-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 具体目标是：（1）通过基于结构的诱变鉴定重要的 DNA连接酶的功能基团;（2）定义 使用足迹法和交联法进行连接酶-腺苷酸和切口DNA; (3)通过晶体学确定连接酶-腺苷酸的结构， DNA切口;和（4）确定连接酶结合的“基态”结构 到ATP。拟议的实验融合了生物化学，分子遗传学， 结构生物学，以阐明化学和构象的步骤， 连接途径是协调的。这些发现将提供新的见解 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的遗传联系 易感性

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.