CRYSTALLOGRAPHIC STUDIES OF DNA REARRANGEMENT ENZYMES

这个子项目是许多利用资源的研究子项目之一 由NIH/NCRR资助的中心拨款提供。子项目的主要支持 而子项目的主要调查员可能是由其他来源提供的， 包括其它NIH来源。 列出的子项目总成本可能 代表子项目使用的中心基础设施的估计数量， 而不是由NCRR赠款提供给子项目或子项目工作人员的直接资金。 DNA重排/重组酶通过切割和重新连接DNA链来改变DNA的共价结构。这些蛋白质在不同的生物环境中发挥重要作用，包括病毒整合到宿主基因组中，维持染色体完整性和产生遗传多样性。使用X射线晶体学，我们正在研究三种不同类型的DNA重排酶，以更好地了解这些酶的功能。 第一类是逆转录病毒整合酶。逆转录病毒，包括引起AIDS的HIV-1，具有在进入宿主细胞后逆转录成线性病毒DNA的RNA基因组。将病毒DNA整合到宿主染色体中是逆转录病毒生命周期中的重要步骤，并由病毒编码的整合酶（IN）蛋白进行。 我们正在利用来自HIV和RSV（Rous肉瘤病毒）系统的IN研究功能性3-结构域IN蛋白以及IN-DNA复合物的晶体结构。 第二类是参与维持细菌线性染色体的DNA解离酶。一些细菌病原体，包括莱姆病螺旋体伯氏疏螺旋体（Borrelia burgdorferi），具有共价闭合发夹末端的线性染色体。这种线性染色体的复制需要将连锁环状中间体分解成单位长度的染色体，这是由发夹形成DNA分解酶进行的。使用疏螺旋体和农杆菌系统，我们正在确定解离酶-DNA复合物的晶体结构，以深入了解DNA链切割和随后的发夹形成的机制。 第三类是参与十字形DNA重组中间体拆分的Holliday连接拆分酶。 我们正在研究与HJ DNA底物复合的痘病毒HJ解离酶的晶体结构。 如果成功，它将是第一个原子结构的酶-DNA复合物的大家庭的HJ resolvases代表的E。coli RuvC.

This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. Primary support for the subproject and the subproject's principal investigator may have been provided by other sources, including other NIH sources. The Total Cost listed for the subproject likely represents the estimated amount of Center infrastructure utilized by the subproject, not direct funding provided by the NCRR grant to the subproject or subproject staff. DNA rearrangement/recombination enzymes alter covalent structure of DNA, by cleaving and rejoining DNA strands. These proteins play important roles in diverse biological contexts, including viral integration into host's genome, maintenance of chromosome integrity, and generation of genetic diversity. Using x-ray crystallography, we are studying three distinct classes of DNA rearrangement enzymes to better understand how these enzymes function. The first class is the retroviral integrase. Retroviruses, including HIV-1 that causes AIDS, have an RNA genome that is reverse-transcribed into a linear viral DNA upon entering the host cell. Integration of this viral DNA into host's chromosome is an essential step in the lifecycle of retroviruses, and is carried out by the virally encoded integrase (IN) protein. We are pursuing crystal structures of the functional 3-domain IN protein as well as IN-DNA complexes, using IN from HIV and RSV (Rous Sarcoma Virus) systems. The second class is the DNA resolvase involved in the maintenance of bacterial linear chromosomes. Some bacterial pathogens, including the Lyme disease spirochete Borrelia burgdorferi, have linear chromosomes with covalently closed hairpin termini. Replication of such linear chromosomes requires resolution of a catenated circular intermediate into unit-length chromosomes, which is carried out by the hairpin-forming DNA resolvase enzyme. Using Borrelia and Agrobacterium systems, we are determining crystal structures of the resolvase-DNA complexes to gain insights into the mechanism of DNA strand cleavages and subsequent hairpin formation. The third class is the Holliday junction resolvase involved in resolution of the cruciform-shaped DNA recombination intermediates. We are pursuing crystal structure of the poxvirus HJ resolvase in complex with HJ DNA substrates. If successful, it will be the first atomic structure of an enzyme-DNA complex for the large family of HJ resolvases represented by E. coli RuvC.