STRUCTURAL STUDIES OF SITE-SPECIFIC RECOMBINATION

描述（由申请人提供）：与噬菌体λ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，包括质粒和染色体的分离、病毒整合、基因表达调控和程序化基因重排。这些酪氨酸重组酶使用酪氨酸侧链催化DNA的切割和连接，而不需要添加高能辅助因子。它们是已知的唯一一种既能产生分支霍利迪连接中间体又能将其分解成重组DNA产物的酶。的DNA重组活性；-int通过与DNA链交换位点两侧的DNA臂位点相互作用来调节。辅助DNA弯曲蛋白压缩DNA底物，使其能够同时相互作用；-int与手臂位点和核心位点的重组。臂位结合相互作用使得化学可逆的重组反应实际上是不可逆的。我们正在开发动力学分析，以测试拟议的机制，为变构控制；通过DNA结合和臂的弯曲来重组酶。小角度x射线散射方法被用来绘制手臂dna缠绕的拓扑结构；复合复合物。作为第二个具有指导意义的例子，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，可以切割dsDNA产生两个发夹端。TelK和相关的端粒分解酶将连接的复制中间体转化为具有共价闭合的发夹末端的单位长度线性染色体。与断裂DNA底物结合的TelK晶体结构表明，蛋白质诱导的DNA畸变使断裂链分离，以防止dsDNA的重新连接，从而形成发夹产物。传统的和单分子的DNA结合、切割、发夹折叠和结扎的测定方法正在开发中。晶体结构将确定额外的TelK-DNA反应中间体。

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.