Chlorella Virus DNA Ligase: Structure and Mechanism

描述(申请人提供)：DNA连接酶是一种普遍存在的酶 催化DNA复制和修复中必不可少的最后一步- 将DNA缺口转化为磷酸二酯键。加入一支5英尺长的队伍 在缺口处从磷酸盐链到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 (连接酶-腺苷)，其中AMP与酶上的赖氨酸相连； AMP从连接酶转移到5‘-磷酸末端，形成 DNA-腺苷中间体；(Iii)连接酶催化3‘-羟基的攻击 缺口DNA-腺苷连接两个多核苷酸，释放AMP。 我们的目标是了解连接酶反应化学是如何被催化的，以及如何 连接酶使用真核病毒DNA识别“受损的”(即，缺口的)DNA 以连接酶为模型。小球藻病毒PBCV1连接酶是最小的真核生物 已知的ATP依赖的连接酶(298-aa)。它只由催化核心组成， 没有装饰细胞连接酶的大的侧翼结构域。 尽管如此，小球藻病毒连接酶维持着有丝分裂的生长和DNA修复 当酵母是细胞中唯一的连接酶时。作为真核生物中最小的连接酶， 还有一种具有固有的缺口传感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 小球藻病毒DNA连接酶的结晶及结构测定 共价连接酶-AMP反应中间体，分辨率为2.0A。 连接酶-AMP结构提出的缺口识别和催化模型 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鉴定重要的 DNA连接酶的功能基团；(2)定义 使用足迹和交联法的连接酶-腺苷和镍DNA； (3)结晶学方法测定连接酶-腺苷酸键的结构 DNA缺口；以及(4)确定连接酶结合的基态结构 为ATP干杯。拟议中的实验融合了生物化学、分子遗传学和 结构生物学，以阐明化学和构象步骤如何 连接途径是协调的。这些发现将提供新的见解 DNA损伤识别--这是一个与人类健康有关的问题 DNA修复途径与人类癌症之间新的遗传联系 性情。

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.