HELICASE CATALYZED DNA UNWINDING

描述(申请人提供)：DNA解旋酶是依赖于三磷酸腺苷的马达蛋白，解离双链DNA，形成DNA代谢所需的单链(Ss)DNA中间体。我们正在研究来自大肠杆菌、Rep、UvrD和RecBCD的三种非六聚体SF1 DNA解旋酶的DNA解离和DNA易位的动力学机制，它们分别在复制、修复和重组中发挥作用。RecBCD是一种含有两个SF1解旋酶(B和D)的杂三聚体复合体。我们的稳态前动力学研究表明，Rep和UvrD解旋酶在体外以寡聚体的形式发挥作用，尽管这些酶的单体可以有效地沿ss-DNA转运。我们在这些单体中发现了一个重要的调控结构域(2B结构域)，当被移除时，它在体外激活了Rep单体的解旋酶活性。虽然2B结构域不是转位所必需的，但2B结构域的大移动与DNA结合和ATP水解有关，因此我们将研究2B结构域在单体单链DNA转位和寡聚体DNA解离中的作用。我们将使用突变和瞬时动力学方法(停流和化学猝灭流)和我们开发的分析方法来检查单体分子马达如何在易位中发挥作用的细节。我们还将测试目前关于寡聚解旋酶复合体如何解开DNA的假设，目的是开发一种完全的动力学机制来解开DNA。SF1解旋酶也可以破坏蛋白质-DNA复合体，我们将研究UvrD破坏RecA-ssDNA细丝的机制。此外，还将对RecBCD和RecBC解旋酶的DNA结合、ATP水解和DNA解离进行机械研究。热力学和动力学研究将被用来理解这些蛋白质如何在依赖于镁而不依赖于ATP的反应中破坏(熔化)DNA碱基对的稳定。我们还将研究辅助蛋白，UvrD的MutL和Rep的PrIC，刺激DNA解离的机制。我们的整体研究将得到这些解旋酶对DNA结合、易位和解离的单分子研究的补充。总体目标是从分子上了解这些分子马达沿脱氧核糖核酸转移和解离的动力学机制(S)，以及这些过程是如何与三磷酸腺苷的结合和水解相耦合的。与公共卫生相关：DNA解旋酶和转位酶在DNA新陈代谢的所有方面都发挥着基础作用，包括在包括人类在内的所有生物体中的DNA复制、重组和修复。一些人类DNA解旋酶的突变与几种人类疾病有关，包括沃纳综合征和布鲁姆综合征。由于它们在核酸代谢中的关键作用，这些酶是可能特异性抑制它们的药物的主要靶点，了解它们的作用机制是至关重要的。

DESCRIPTION (provided by applicant): DNA helicases are ATP-dependent motor proteins that unwind duplex DNA to form the single stranded (ss) DNA intermediates required for DNA metabolism in all organisms. We are studying the kinetic mechanisms of DNA unwinding and DNA translocation of three non-hexameric SF1 DNA helicases from E. coli, Rep, UvrD, and RecBCD, which function in replication, repair, and recombination, respectively. RecBCD is a hetero-trimeric complex containing two SF1 helicases (B and D). Our pre-steady state kinetic studies indicate that Rep and UvrD helicases function as oligomers in vitro, even though monomers of these enzymes can translocate efficiently along ss-DNA. We have discovered an important regulatory domain within these monomers (2B domain) that, when removed, activates helicase activity of a Rep monomer in vitro. Although the 2B domain is not needed for translocation, large movements of the 2B domain are coupled to DNA binding and ATP hydrolysis, hence we will study the role of the 2B domain in monomer ssDNA translocation and DNA unwinding by oligomers. We will use mutagenesis and transient kinetic approaches (stopped-flow and chemical quenched-flow) and methods of analysis that we have developed to examine the details of how the monomeric molecular motor functions in translocation. We will also test current hypothesis for how the oligomeric helicase complexes unwind DNA, with the goal of developing a full kinetic mechanism for unwinding. SF1 helicases also function to disrupt protein-DNA complexes, and we will study the mechanism by which UvrD disrupts RecA-ssDNA filaments. DNA binding, ATP hydrolysis and DNA unwinding by RecBCD and RecBC helicases will also be examined mechanistically. Thermodynamic and kinetic studies will be used to understand how these proteins destabilize (melt) DNA base pairs in a Mg2+dependent, but ATP-independent reaction. We will also examine the mechanism by which accessory proteins, MutL for UvrD and PriC for Rep, stimulate DNA unwinding. Our ensemble studies will be complemented by single molecule studies of DNA binding, translocation and unwinding by these helicases. The overall goal is to obtain a molecular understanding of the kinetic mechanism(s) by which these molecular motors translocate along and unwind DNA and how these processes are coupled to ATP binding and hydrolysis. PUBLIC HEALTH RELEVANCE: DNA helicases and translocases play fundamental roles in all aspects of DNA metabolism, including DNA replication, recombination and repair in all organisms including humans. Mutations in a number of human DNA helicases are linked to several human diseases, including Werner's and Bloom's syndromes. Because of their pivotal roles in nucleic acid metabolism, these enzymes are prime targets for drugs that may inhibit them specifically, and it is critical to understand their mechanisms of action.