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分子马达蛋白，可解离双链DNA，形成DNA代谢和基因组维持所需的单链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单位DNA解旋酶/核酸酶的DNA解离和单链DNA移位的机制，该酶在DNA双链断裂和重组的修复中发挥作用。RecBCD是一种含有两个超家族1(SF1)解旋酶/转位酶马达(RecB，3‘到5’马达和RecD，5‘到3’马达)的异三聚体复合体，它们以不同的速度运动，虽然是同一复合体的一部分，但在RecC亚基识别出一个8核苷酸的单链DNA序列后，相对速率发生了转换，称为“chi”。由于CHI识别的变构效应，RecBCD的核酸酶活性也发生了显着变化。虽然人们对解旋酶已经有了很多了解，但对解开DNA的基本机制(S)仍然知之甚少。关于这两个马达如何在像RecBCD这样的解旋酶中进行交流也鲜为人知。我们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)还具有以前未被发现的次要转位酶活性，该活性可使RecBC沿相反的DNA链移动。因此，一个RecB马达驱动两个转位酶活动。我们的目标是：1-确定二级RecBC转位酶在RecBC中的位置以及它是否在RecBCD内发挥功能；2-使用我们新的荧光分析来确定RecB和RecD马达在RecBCD解链DNA过程中是否进行通信，并测试我们的假设，即二级转位酶活性在两个运动亚单位(RecB和RecD)之间的通信中发挥功能作用，以及它们被chi调控；3-确定RecBCD和RecBC可以在镁离子依赖但不依赖于ATP的过程中从钝化的DNA中融化4-6个碱基的机制；4-确定在DNA解旋酶活性过程中，DNA融化是否独立于ssDNA转位发生。将使用热力学、瞬时动力学、结构和单分子方法(荧光和光钳)来获得对这些分子马达沿DNA移位和解离DNA的动力学机制(S)的分子理解，以及这种多组分解旋酶是如何被调节的。这样的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。

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.