HELICASE CATALYZED DNA UNWINDING

描述（由申请人提供）：DNA解旋酶是atp依赖的运动蛋白，它解开双链DNA形成所有生物体DNA代谢所需的单链（ss） DNA中间体。我们正在研究大肠杆菌Rep、UvrD和RecBCD三种非六聚体SF1 DNA解旋酶的DNA解绕和DNA易位的动力学机制，这三种酶分别在复制、修复和重组中起作用。RecBCD是一种含有两个SF1解旋酶（B和D）的异三聚物。我们的稳态前动力学研究表明，Rep和UvrD解旋酶在体外作为低聚物发挥作用，尽管这些酶的单体可以沿着ss-DNA有效地转运。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），当它被移除时，在体外激活Rep单体的解旋酶活性。虽然2B结构域不需要易位，但2B结构域的大运动与DNA结合和ATP水解有关，因此我们将研究2B结构域在单体ssDNA易位和DNA低聚物解绕中的作用。我们将使用诱变和瞬态动力学方法（停止流动和化学猝灭流动）以及我们开发的分析方法来检查单体分子运动在易位中的功能细节。我们还将测试目前关于寡聚解旋酶复合物如何解开DNA的假设，目的是开发一个完整的解旋动力学机制。SF1解旋酶也有破坏蛋白质- dna复合物的功能，我们将研究UvrD破坏RecA-ssDNA细丝的机制。RecBCD和RecBC解旋酶的DNA结合、ATP水解和DNA解绕也将进行机理研究。热力学和动力学研究将用于了解这些蛋白质如何在Mg2+依赖但atp独立的反应中破坏（融化）DNA碱基对。我们还将研究辅助蛋白（UvrD的MutL和Rep的pricc）刺激DNA解绕的机制。我们的整体研究将由这些解旋酶的DNA结合、易位和解绕的单分子研究来补充。总体目标是获得这些分子马达沿DNA移动和展开的动力学机制的分子理解，以及这些过程如何与ATP结合和水解耦合。公共卫生相关性：DNA解旋酶和转位酶在包括人类在内的所有生物体的DNA代谢的所有方面，包括DNA复制、重组和修复中发挥着基本作用。一些人类DNA解旋酶的突变与几种人类疾病有关，包括维尔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至关重要。

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.