HELICASE CATALYZED DNA UNWINDING

描述（由申请人提供）：DNA解旋酶是ATP依赖性分子马达蛋白，其解旋双链DNA以形成所有生物体中DNA代谢和基因组维持所需的单链（ss）DNA中间体。DNA解旋酶的缺陷是许多人类疾病的原因。我们研究了多亚基DNA解旋酶/核酸酶E. coliRecBCD，其功能是修复DNA双链断裂和重组。RecBCD是一种异源三聚体复合物，含有两个超家族1（SF 1）解旋酶/转位酶马达（RecB，3'至5'马达和RecD，5'至3'马达），它们以不同的速率移动，同时是同一复合物的一部分，但在RecC亚基识别8个核苷酸的ssDNA序列（称为“chi”）后，相对速率发生转换。RecBCD的核酸酶活性也由于chi识别的变构效应而显著改变。尽管人们对解旋酶了解很多，但对DNA解旋的基本机制仍然知之甚少。关于这两个马达如何在像RecBCD这样的解旋酶中交流也知之甚少。我们已经开发了一种新的荧光测定法，使我们能够发现，除了其主要的3'至5'易位酶，RecBC（无RecD）还具有以前未被认识到的次级易位酶活性，该活性使RecBC沿着相反的DNA链移动。因此，一个RecB马达驱动两个转位酶活性。我们的目标是：1-确定次级RecBC移位酶在RecBC内的位置以及它是否在RecBCD内起作用; 2-使用我们的新型荧光测定法来确定RecB和RecD马达是否在RecBCD的DNA解旋期间进行通信，并测试我们的假设，即次级移位酶活性在两个马达亚基之间的通信中起功能性作用（RecB和RecD）和它们通过chi的调节; 3-确定RecBCD和RecBC可以在Mg 2+依赖性但不依赖于ATP的过程中从平端DNA中熔解掉4-6 bp的机制，以及4-确定在DNA解旋酶活性期间DNA熔解是否与ssDNA易位分开发生。热力学，瞬态动力学，结构和单分子的方法（荧光和光镊）将被用来获得动力学机制（S）的分子理解，这些分子马达易位沿着和解开DNA，以及如何调节这种多组分解旋酶。这些研究将为这些核酸马达酶提供新的见解，这些酶对维持所有基因组至关重要。 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。由于它们在包括细菌在内的所有生物体的核酸代谢中发挥着关键作用，这些酶是可能特异性抑制它们的药物或抗生素的主要靶点，因此了解它们的作用机制非常重要。

DESCRIPTION (provided by applicant): DNA helicases are ATP-dependent molecular motor proteins that unwind duplex DNA to form the single stranded (ss) DNA intermediates required for DNA metabolism and genome maintenance in all organisms. Defects in DNA helicases are responsible for a number of human diseases. We are studying the mechanisms of DNA unwinding and ssDNA translocation of a multi-subunit DNA helicase/nuclease, E. coli RecBCD, which functions in repair of DNA double strand breaks and recombination. RecBCD is a hetero-trimeric complex containing two superfamily 1 (SF1) helicase/translocase motors (RecB, a 3' to 5' motor and RecD, a 5' to 3' motor) that move with different rates while part of the same complex, but undergo a switch in relative rates after the RecC subunit recognizes an 8 nucleotide ssDNA sequence, called "chi". The nuclease activity of RecBCD is also changed dramatically due to an allosteric effect of chi recognition. Although much is known about helicases, the basic mechanism(s) of DNA unwinding is still poorly understood. There is also little known about how the two motors might communicate within a helicase like RecBCD. We have developed a novel fluorescence assay that enabled us to discover that, in addition to its primary 3' to 5' translocase, RecBC (without RecD) also possesses a previously unrecognized secondary translocase activity that moves RecBC along the opposite DNA strand. Hence, the one RecB motor drives two translocase activities. Our goals are to: 1- determine the location of the secondary RecBC translocase within RecBC and if it functions within RecBCD; 2- use our novel fluorescence assay to determine whether the RecB and RecD motors communicate during DNA unwinding by RecBCD and test our hypothesis that the secondary translocase activity plays a functional role in the communication between the two motor subunits (RecB and RecD) and their regulation by chi; 3- determine the mechanism by which RecBCD and RecBC can melt out 4-6 bp from a blunt ended DNA in a Mg2+- dependent, but ATP-independent process and, 4- determine if DNA melting occurs separately from ssDNA translocation during DNA helicase activity. Thermodynamic, transient kinetic, structural and single molecule approaches (fluorescence and optical tweezers) will be used to obtain a molecular understanding of the kinetic mechanism(s) by which these molecular motors translocate along and unwind DNA and of how this multi-component helicase is regulated. Such studies will provide new insight into these nucleic acid motor enzymes that are essential for the maintenance of all genomes. 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 in all organisms, including bacteria, these enzymes are prime targets for drugs or antibiotics that may inhibit them specifically, and it is therefore important o understand their mechanisms of action.