HELICASE CATALYZED DNA UNWINDING

描述（由申请人提供）：DNA解旋酶是ATP依赖性马达蛋白，其解旋双链体DNA以形成所有生物体中DNA代谢所需的单链（ss）DNA中间体。我们研究了大肠杆菌三种非六聚体SF 1 DNA解旋酶的DNA解旋和DNA转位的动力学机制。coli、Rep、UvrD和RecBCD，它们分别在复制、修复和重组中起作用。RecBCD是含有两个SF 1解旋酶（B和D）的异源三聚体复合物。我们的预稳态动力学研究表明，Rep和UvrD解旋酶的功能作为低聚体在体外，即使这些酶的单体可以有效地沿沿着ss-DNA易位。我们已经发现了一个重要的调节结构域在这些单体（2B域），当删除，激活解旋酶活性的Rep单体在体外。虽然2B结构域不需要易位，但2B结构域的大移动与DNA结合和ATP水解偶联，因此我们将研究2B结构域在单体ssDNA易位和寡聚体DNA解旋中的作用。我们将使用诱变和瞬时动力学方法（停流和化学猝灭流）和我们已经开发的分析方法来研究单体分子马达如何在易位中发挥作用的细节。我们还将测试目前的假设，寡聚解旋酶复合物如何解开DNA，与发展一个完整的动力学机制解旋的目标。SF 1解旋酶也可以破坏蛋白质-DNA复合物，我们将研究UvrD破坏RecA-ssDNA丝的机制。DNA结合，ATP水解和DNA解旋的RecBCD和RecBC解旋酶也将进行机械检查。热力学和动力学研究将用于了解这些蛋白质如何在Mg 2+依赖性但不依赖于ATP的反应中使DNA碱基对不稳定（熔化）。我们还将研究辅助蛋白，MutL为UvrD和PriC为Rep，刺激DNA解旋的机制。我们的合奏研究将补充这些解旋酶的DNA结合，易位和解旋的单分子研究。总体目标是获得对动力学机制的分子理解，通过该机制这些分子马达沿沿着和展开DNA移位，以及这些过程如何与ATP结合和水解偶联。 公共卫生相关性：DNA解旋酶和转位酶在DNA代谢的各个方面发挥着重要作用，包括DNA复制，重组和修复，在所有生物体中，包括人类。许多人类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卢姆综合征。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.