CRYSTALLOGRAPHIC STUDIES OF DNA REARRANGEMENT ENZYMES

这个子项目是许多研究子项目中的一个 由NIH/NCRR资助的中心赠款提供的资源。子项目及 研究者（PI）可能从另一个NIH来源获得了主要资金， 因此可以在其他CRISP条目中表示。所列机构为 研究中心，而研究中心不一定是研究者所在的机构。 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学背景中发挥重要作用，包括病毒整合到宿主基因组中和维持染色体完整性。利用X射线晶体学，我们正在研究两种不同的DNA重排酶与人类疾病有关。 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。尽管逆转录病毒IN具有高度的医学相关性，但对于负责两个病毒DNA末端协同整合的完整3结构域IN蛋白，没有结构信息可用。我们正在使用的功能3域IN蛋白质的晶体结构，迫切需要更好地了解IN催化反应的机理。以劳斯肉瘤病毒（RSV）为模型系统，我们获得了具有衍射质量的三结构域IN晶体。需要射束时间进行更高分辨率的数据收集和定相实验。 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，以更好地理解DNA链切割和随后的发夹形成的机制。

This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. 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 and maintenance of chromosome integrity. Using x-ray crystallography, we are studying two distinct classes of DNA rearrangement enzymes relevant to human diseases. 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. Despite high medical relevance of retroviral IN, no structural information is available for the intact 3-domain IN protein responsible for the concerted integration of two viral DNA ends. We are using crystal structures of the functional 3-domain IN protein critically needed for a better mechanistic understanding of IN-catalyzed reactions. Using Rous Sarcoma Virus (RSV) as a model system, we have obtained a diffraction-quality crystal of 3-domain IN. Beam time is requested for higher resolution data collection and phasing experiments. 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 better understand the mechanism of DNA strand cleavages and subsequent hairpin formation.