Enzymatic Mechanisms of DNA Helicases

描述（由申请人提供）：DNA解旋酶提供了将双链体DNA转化为单链DNA（ssDNA）的主要机制，用作DNA复制和修复中的模板或重组中的底物。事实上，这些酶对于DNA复制、修复和维持所有生物体的基因组稳定性至关重要。与这一观点相一致的是，人类细胞中编码DNA解旋酶的基因缺陷与基因组不稳定性有关，导致各种早老性疾病和人类癌症。因此，完全理解这些酶的机制和作用对于理解细胞内DNA交易的基本原理至关重要。在上一个资助期间，我们在理解错配修复蛋白MutL和UvrD之间的相互作用方面取得了重大进展。这种相互作用的结果在一个显着的刺激UVRD催化解旋。我们已经表明，MutL有助于加载UvrD，MutL必须有其ATP辅因子结合，以刺激UvrD，我们现在有初步的结果表明，MutL作为一个持续合成因子，以增加UvrD解绕的双链DNA的长度。当目标1中提出的动力学和生物物理实验完全证实时，这将是解旋酶持续合成因子的第一个很好描述的例子。我们还解剖了F质粒上traI基因编码的DNA解旋酶I的结构域。我们已经证明，这种解旋酶作为单体是活跃的，具有非常强的进行性和非常快的速度。我们将使用单分子实验来直接证明目标2中提出的蛋白质的持续合成能力。我们还将使用生物化学，生物物理和结构实验来定义这种蛋白质催化进行性解旋的机制。这代表了一个独特的机会，了解单体蛋白质如何实现显着的持续合成能力。最后，我们最近表明，UvrD解开霍利迪结基板，大概是在交界处开始。我们假设UvrD的作用是破坏不需要的重组中间体，其中包含涉及MutS和MutL的途径中的错配，并且对于确保基因组稳定性至关重要。在目标3中，将使用动力学和生物化学实验来研究UvrD的这种新反应和生物学作用。公共卫生相关性：DNA解旋酶对于DNA复制、修复、重组和维持所有生物体的基因组稳定性至关重要。由于发现特定DNA解旋酶的突变会导致几种人类遗传疾病，人们对充分了解这类蛋白质的兴趣得到了提高。随着更多的疾病基因被鉴定出来，理解这类蛋白质的重要性就更加突出了，其他疾病很可能与DNA解旋酶的缺陷有关。

DESCRIPTION (provided by applicant): DNA helicases provide the primary mechanism by which duplex DNA is converted to single-stranded DNA (ssDNA) for use as a template in DNA replication and repair or as a substrate in recombination. Indeed, these enzymes are essential for DNA replication, repair and to maintain genomic stability in all organisms. Consistent with this idea, defects in genes encoding DNA helicases in human cells have been linked to genomic instability leading to a variety of progeriod disorders and human cancers. Thus, a complete understanding of the mechanisms and roles played by these enzymes is essential to understanding the fundamentals of DNA transactions within the cell. During the last grant period we have made significant progress in understanding the interaction between a mismatch repair protein, MutL, and UvrD. This interaction results in a dramatic stimulation of UvrD-catalyzed unwinding. We have shown that MutL helps load UvrD, that MutL must have its ATP cofactor bound to stimulate UvrD and we now have preliminary results indicating that MutL acts as a processivity factor to increase the length of duplex DNA unwound by UvrD. When fully confirmed by the kinetic and biophysical experiments proposed in aim 1, this will be the first well-described example of a helicase processivity factor. We have also dissected the domain structure of DNA helicase I encoded by the traI gene on the F plasmid. We have shown this helicase to be active as a momomer, extraordinarily processive and very fast. We will use single molecule experiments to directly demonstrate the processivity of the protein as proposed in aim 2. We will also use biochemical, biophysical and structural experiments to define the mechanism by which this protein catalyzes processive unwinding. This represents a unique opportunity to understand how a monomeric protein can achieve remarkable processivity. Finally, we have recently shown that UvrD unwinds Holliday Junction substrates, presumably initiating at the junction. We hypothesize that UvrD acts to destroy unwanted recombination intermediates containing a mismatch in a pathway that involves MutS and MutL, and is essential to ensure genomic stability. This new reaction and biological role for UvrD will be investigated using kinetic and biochemical experiments in aim 3. PUBLIC HEALTH RELEVANCE: DNA helicases are essential for DNA replication, repair, recombination and to maintain genomic stability in all organisms. Interest in fully understanding this class of proteins has been heightened by the discovery that mutations in specific DNA helicases result in several human genetic diseases. It is likely that other diseases will be linked with defects in DNA helicases as more disease genes are identified placing added emphasis on the importance of understanding this class of proteins.