MOLECULAR MECHANISM OF THE VMA1 DERIVED ENDONUCLEASE

VMA1 衍生核酸内切酶的分子机制

基本信息

批准号：
2701614
负责人：
FREDERICK Samuel GIMBLE
金额：
$ 10.87万
依托单位：
TEXAS A&M AGRILIFE RESEARCH
依托单位国家：
美国
项目类别：
财政年份：
1994
资助国家：
美国
起止时间：
1994-05-01 至 1999-04-30
项目状态：
已结题

项目摘要

The mapping and manipulation of complex genomes will be greatly facilitated by the availability of reagents that cleave DNA at sites that occur infrequently. Such reagents will also be useful for gene targeting projects that am to correct the inborn mutations that cause genetic disease. A family of related DNA endonucleases has been identified that will accelerate these studies. A common feature of these enzymes is that they initiate genomic rearrangement events within their host organisms by making double-stranded breaks at specific sites. The molecular mechanism of these enzymes is unknown. However, the conservation of structural features in this family, including the presence of two dodecapeptide motifs, suggests that information learned about the structure and function of one enzyme will be directly applicable to the other members of the family. The objective of the proposed research is to understand how the structural and mechanistic features of one such endonuclease from yeast, called VDE, results in site-specific cleavage. The VDE endonuclease has been purified to homogeneity as an active enzyme and its substrate has been identified. The first aim is to determine whether VDE cleaves DNA by a concerted or a sequential reaction mechanism and to ascertain the oligomeric form of the enzyme during cleavage. Kinetic parameters for VDE-mediated cleavage will be measured under a variety of conditions using cognate and non-cognate substrates to reveal the reaction mechanism. The oligomeric state of the enzyme will be deduced from gel-shift experiments. The second goal is to assess the relative importance of the base-pairs within the recognition site in the DNA cleavage reaction. The recognition site will be randomly mutagenized, and these substrates will be tested for VDE binding and cleavage activities. An optimized cleavage/recognition site will be generated by a novel PCR-based technique. The third aim is to determine which amino acids within the dodecapeptide motifs and elsewhere in the protein affect catalytic function and determine specificity. Mutant proteins generated by site-directed and cassette mutagenesis will be assayed for catalytic and DNA-binding function and the mutations will be located by sequence analysis. Chimeric proteins will be constructed between VDE and the related HO endonuclease in order to localize the specificity determinants. In the long term we would like to use this data to alter substrate specificity.

复杂基因组的映射和操纵将是极大的通过在裂解DNA的试剂的可用性来促进很少发生。这样的试剂也对基因靶向有用纠正导致遗传的天生突变的项目疾病。已经确定了一个相关的DNA核酸内切酶的家族将加速这些研究。这些酶的一个共同特征是他们通过其宿主生物体中的基因组重排事件在特定地点进行双链休息。分子机制这些酶是未知的。但是，结构的保护这个家庭的特征，包括两个十二肽的存在主题表明，有关结构和功能的信息一个酶的一个将直接适用于其他成员家庭。拟议研究的目的是了解结构如何和一种此类核酸酶的机械特征，来自酵母，称为VDE，导致特定地点的裂解。 VDE核酸内切酶已被纯化已经确定了作为活性酶及其底物的同质性。第一个目的是确定VDE是通过一致还是一致的裂解DNA 顺序反应机制，并确定寡聚形式裂解过程中的酶。 VDE介导的裂解的动力学参数将在多种条件下使用同名和非同在底物揭示了反应机制。寡聚状态酶将从凝胶转移实验中推导。第二个目标是评估基准对在识别中的相对重要性位点在DNA裂解反应中。识别站点将是随机的诱变，这些底物将进行VDE结合和切割活动。优化的分裂/识别站点将是由新型基于PCR的技术生成。第三个目的是确定十二肽基序中的氨基酸以及在其他地方的其他地方蛋白质会影响催化功能并确定特异性。突变体由位置定向和盒式诱变产生的蛋白质将是测定催化和DNA结合功能，突变将是位于序列分析。将构建嵌合蛋白在VDE和相关的HO核酸内切酶之间，以本地化特异性决定因素。从长远来看，我们想使用此数据改变底物特异性。