Structural studies of DNA resolvases involved in hairpin telomere maintenance

描述(申请人提供)：端粒保护线性染色体的末端，促进末端DNA序列的完全复制。端粒最简单的形式是一种共价闭合的发夹结构，在细菌和病毒中发现，这些病毒携带线性染色体，包括疏螺旋体属的成员--莱姆病和复发性发烧的病原体--以及痘病毒。带有发夹端粒的线性染色体的复制通过两步机制进行，在该机制中，DNA聚合酶首先产生串联的复制中间体，然后将其分解成单位长度的染色体。这项提议的重点是细菌和痘病毒酶，它可以分解连体染色体来再生发夹端粒。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副本的反向重复DNA序列。细菌原端粒酶利用光焦氨酸介导的DNA裂解-再连接反应将回文双链底物分解成发夹状产物。另一方面，痘病毒裂解酶以回文序列与发夹挤出形成的Holliday连接结构特异性结合，并在连接点上进行对称链切割。尽管细菌和痘病毒DNA解析酶催化的反应的化学性质已经很好地确定了，但这些蛋白质如何适应各自的催化模块来在复制的端粒位置上解析DNA仍然知之甚少。在这项提议中，我们将通过确定各种解旋酶-DNA复合体的晶体结构来具体解决以下问题：细菌原端粒酶如何利用固有的等能量DNA裂解-重新连接化学促进双重底物有效地重新折叠成发夹产品？痘病毒解析酶是如何在识别分支DNA结构和催化连接点的协同链切割方面实现高度特异性的？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分解酶在跨反向重复连接进行对称DNA切割方面可能有相似的策略。我们的结构工作将突出不同的策略以及潜在的酶在重要病原体中维持发夹端粒所使用的一般机制。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拆分酶具有相似反应化学性质的DNA重排机制。 与公共卫生相关：我们将调查基因信息是如何在某些导致人类疾病或食品工业问题的细菌和病毒中保持和复制的。通过这项研究获得的信息将有助于药物的设计，并可能有助于开发在广泛应用中操纵DNA的工具。

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.