Structural studies of DNA resolvases involved in hairpin telomere maintenance

描述（由申请人提供）：端粒保护线性染色体的末端，促进末端DNA序列的完整复制。端粒最简单的形式是在携带线性染色体的细菌和病毒中发现的共价闭合发夹结构，包括伯氏疏螺旋体属成员——莱姆病和回归热的病原体——和痘病毒。具有发夹端粒的线性染色体的复制通过两步机制进行，其中DNA聚合酶首先产生连接的复制中间体，随后分解成单位长度的染色体。这一建议的重点是细菌和痘病毒酶解决连接染色体再生发夹端粒。这两类DNA分解酶利用不同类型的化学反应来处理分离多个染色体拷贝的反向重复DNA序列。细菌前端粒酶利用磷酪氨酸介导的DNA切割-再连接反应将回文双相底物分解成发夹产物。另一方面，痘病毒分解酶在一个回文序列上特异性地结合由发夹挤压形成的Holliday结结构，并在结点上形成对称的链切割。尽管由细菌和病毒DNA分解酶催化的反应的化学性质已经很好地确定，但人们对这些蛋白质如何适应各自的催化模块来在复制的端粒位点上分解DNA知之甚少。在本提案中，我们将通过确定各种分解- DNA复合物的晶体结构来具体解决以下问题：细菌前端粒酶如何利用内在等能DNA切割-再连接化学促进双相底物有效地折叠成发夹产物？痘病毒如何在识别支链DNA结构和在连接点催化协调链切割方面达到高特异性？尽管在生化水平上进行的反应似乎彼此独立，但这两种类型的DNA分解酶在反向重复连接上产生对称的DNA切割时可能具有相似的策略。我们的结构工作将突出不同的策略，以及潜在的一般机制所采用的酶参与维护发夹端粒在重要的病原体。此外，我们的研究可能有助于更好地理解许多与本文研究的DNA分解酶具有相似反应化学的DNA重排机制。

DESCRIPTION (provided by applicant): Telomeres protect the ends of a linear chromosome and facilitate complete replication of terminal DNA sequences. The simplest form of telomere is a covalently closed hairpin structure found in bacteria and viruses carrying linear chromosomes, including members of the genus Borrelia - the causative agents of Lyme disease and relapsing fever - and the poxviruses. Replication of a linear chromosome with hairpin telomeres proceeds through a two-step mechanism, in which DNA polymerases first produce a concatenated replication intermediate that is subsequently resolved into unit-length chromosomes. This proposal focuses on the bacterial and poxviral enzymes that resolve the concatemeric chromosome to regenerate hairpin telomeres. The two classes of DNA resolvases utilize distinct types of chemical reactions to process the inverted repeat DNA sequences separating multiple copies of chromosomes. Bacterial protelomerases resolve a palindromic duplex substrate into hairpin products using the phophotyrosine-mediated DNA cleavage-rejoining reaction. On the other hand, the poxvirus resolvase binds specifically to the Holliday junction structure formed by hairpin extrusion at a palindromic sequence and makes symmetrical strand cleavages across the junction point. Even though the chemical natures of the reactions catalyzed by the bacterial and poxviral DNA resolvases are well established, it is poorly understood how these proteins adapt the respective catalytic modules to resolve DNA at the sites of replicated telomeres. In this proposal we will specifically address the following questions by determining crystal structures of various resolvase- DNA complexes: How do the bacterial protelomerase enzymes facilitate efficient refolding of a duplex substrate into hairpin products using the intrinsically isoenergetic DNA cleavage-rejoining chemistry? How does the poxvirus resolvase achieve high specificity in recognizing the branched DNA structure and catalyzing concerted strand cleavages at the junction point? Despite carrying out reactions seemingly independent of each other at the biochemical level, the two types of DNA resolvases may share a similar strategy in making symmetrical DNA cleavages across the inverted repeat junction. Our structural work will highlight diverse strategies as well as potentially a general mechanism employed by enzymes involved in the maintenance of hairpin telomeres in the important pathogens. Furthermore, our research may contribute to better understanding of many DNA rearrangement machineries that share similar reaction chemistries with the DNA resolvases studied here. PUBLIC HEALTH RELEVANCE: We will investigate how the genetic information is maintained and replicated in certain bacteria and viruses that cause human diseases or problems in food industry. The information obtained through this research will help design drugs, and may contribute to development of tools for manipulating DNA in a wide range of applications.