STRUCTURAL STUDIES OF SITE-SPECIFIC RECOMBINATION

描述(申请人提供)：与噬菌体lambda整合酶蛋白(；-int)相关的位点特异性DNA重组酶具有多种生物学功能，包括分离质粒和染色体、病毒整合、基因表达调节和程序性基因重排。这些酪氨酸重组酶利用酪氨酸侧链催化DNA切割和连接，而不需要添加高能辅因子。它们是已知的唯一既能产生分支Holliday连接中间体又能将它们分解成重组DNA产物的酶。-INT的DNA重组活性受与DNA链交换部位两侧的DNA臂部位相互作用的调节。辅助DNA弯曲蛋白使DNA底物紧凑，从而能够同时与臂部位和重组的核心部位相互作用。臂-位相互作用使化学上可逆的复合反应有效地不可逆。我们正在开发动力学分析来测试所提出的通过DNA结合和手臂弯曲来控制；-int重组酶变构的机制。小角X射线散射法被用来绘制包裹在臂DNA上的拓扑结构；重组复合体。作为第二个有启发性的例子，我们正在研究相关的telk蛋白，这是一种重要的噬菌体复制蛋白，它能切割dsDNA产生两个发夹末端。TELK和相关的端粒裂解酶将串联的复制中间产物转化为单位长度的线性染色体，末端为共价闭合的发夹。与切割的DNA底物结合的TELK的晶体结构表明，蛋白质诱导的DNA扭曲分离了切割的链，以防止dsDNA的重新连接，从而能够形成发夹产物。DNA结合、切割、发夹折叠和连接的常规和单分子分析正在开发中。其他Telk-DNA反应中间体的晶体结构将被确定。 酪氨酸重组酶和TELK催化类似的化学反应，对抗起始双链DNA底物(S)的扭转刚性和热力学稳定性，以重塑DNA。-int和telk通过酶-DNA复合体的不同结构及其对DNA断裂链定位的影响而实现不同的结果。我们对这些酶的结构-功能的联合分析将使我们更深入地理解蛋白质诱导的DNA结构扭曲与磷酸二酯键断裂和形成之间的能量耦合。这一知识作为一种基因传递技术，在改进定点重组方面具有应用价值。与公共卫生相关：我们正在研究切割和重新连接DNA的酶，在细菌和病毒中执行基本任务。这些酶具有潜在的基因工程应用，可以在人类基因组的精确位置插入基因。它们也是开发杀死细菌的新抗生素的潜在目标。

DESCRIPTION (provided by applicant): Site-specific DNA recombinases related to the phage lambda integrase protein (;-int) have diverse biological functions including the segregation of plasmids and chromosomes, viral integration, regulation of gene expression, and programmed gene rearrangements. These tyrosine recombinases catalyze DNA cleavage and ligation using a tyrosine side chain without the addition of a high energy cofactor. They are the only enzymes known to both create branched Holliday junction intermediates and to resolve them into recombinant DNA products. The DNA recombination activity of ;-int is regulated by interactions with DNA arm sites flanking the site of DNA strand exchange. Accessory DNA bending proteins compact the DNA substrate to enable simultaneous interactions of ;-int with arm-sites and the core-sites of recombination. Arm-site binding interactions make the chemically reversible recombination reaction effectively irreversible. We are developing kinetic assays to test a proposed mechanism for the allosteric control of the ;-int recombinase by DNA binding and bending of the arms. Small angle x-ray scattering methods are being used to map the topology of the arm DNAs wrapping around ; recombination complexes. As a second instructive example, we are studying the related TelK protein, an essential phage replication protein that cleaves dsDNA to generate two hairpin ends. TelK and related telomere resolvases convert concatenated replication intermediates into unit length linear chromosomes with covalently-closed, hairpin ends. Crystal structures of TelK bound to cleaved DNA substrates suggest that a protein-induced distortion of DNA separates the cleaved strands to prevent re-ligation of the dsDNA, enabling formation of hairpin product. Conventional and single molecule assays of DNA binding, cleavage, hairpin folding, and ligation are being developed. Crystal structures will be determined of additional TelK-DNA reaction intermediates. The tyrosine recombinases and TelK catalyze similar chemical reactions, working against the torsional stiffness and thermodynamic stability of the starting double-stranded DNA substrate(s) in order to remodel the DNA. Different outcomes by ;-int and TelK are achieved through different architectures of the enzyme-DNA complexes and their effects on positioning the cleaved strands of DNA. Our combined structure-function analyses of these enzymes will provide a deeper understanding of the energetic coupling between protein-induced distortions of DNA structure and the chemistry of phosphodiester bond cleavage and formation. This knowledge has application in improving site-specific recombination as a gene delivery technology. PUBLIC HEALTH RELEVANCE: We are studying enzymes that cut and rejoin DNA, performing essential tasks in bacteria and viruses. These enzymes have potential genetic engineering applications for inserting genes at precise locations of the human genome. They are also a potential target for the development of new antibiotics that kill bacteria.