HELICASE CATALYZED DNA UNWINDING

描述（由申请人提供）：DNA解旋酶是atp依赖的分子马达蛋白，在所有生物体中解开双链DNA形成DNA代谢和基因组维持所需的单链（ss） DNA中间体。DNA解旋酶的缺陷是导致许多人类疾病的原因。我们正在研究一种多亚基DNA解旋酶/核酸酶，大肠杆菌RecBCD的DNA解绕和ssDNA易位的机制，该酶在DNA双链断裂的修复和重组中起作用。RecBCD是一种异三聚体复合体，包含两个超家族1 （SF1）解旋酶/转位酶马达（RecB，一个3‘到5’马达和RecD，一个5‘到3’马达），它们在相同复合体的一部分以不同的速度移动，但在RecC亚基识别8个核苷酸的ssDNA序列后，发生相对速率的转换，称为“chi”。由于chi识别的变构效应，RecBCD的核酸酶活性也发生了巨大的变化。尽管人们对解旋酶了解甚多，但对DNA解绕的基本机制仍知之甚少。关于这两个马达如何在像RecBCD这样的解旋酶内进行通信也知之甚少。我们已经开发了一种新的荧光分析方法，使我们能够发现，除了其主要的3‘到5’转位酶外，RecBC（不含RecD）还具有以前未被识别的次级转位酶活性，该活性使RecBC沿着相反的DNA链移动。因此，一个RecB马达驱动两个易位活动。我们的目标是：1-确定次级RecBC易位在RecBC中的位置，以及它是否在RecBCD中起作用；2-利用我们的新荧光法确定RecBCD在DNA解绕过程中RecB和RecD马达是否通信，并验证我们的假设，即次级转座酶活性在两个马达亚基（RecB和RecD）之间的通信以及chi对它们的调节中起功能作用；3-确定RecBCD和RecBC在Mg2+依赖但不依赖于atp的过程中从钝端DNA熔化4-6 bp的机制；4-确定DNA解旋酶活性期间DNA熔化是否与ssDNA易位分开发生。热力学、瞬态动力学、结构和单分子方法（荧光和光学镊子）将被用来获得这些分子马达沿着DNA移位和展开的动力学机制的分子理解，以及这种多组分解旋酶是如何被调节的。这些研究将为这些对所有基因组的维持至关重要的核酸运动酶提供新的见解。

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.