BIOCHEMICAL MECHANISM AND FUNCTION OF DNA HELICASES

DNA解旋酶代表了一类普遍存在的核酸酶。 这些蛋白质将dsDNA“解离”(即变性)为单独的单链DNA. DNA链，在需要核苷三磷酸的反应中 水解液。这项建议的总体目标是理解 DNA解旋酶作用的生化机制和生物学功能， 即解旋酶是如何沿着单链DNA(SsDNA)转运到 解开双链(DsDNA)，在这样做之后，这一努力是如何进行的 转化为生物功能(即有用的工作)。一个 理解这些根本问题将有助于我们 了解这些解旋酶的功能和转位 蛋白质，大体上。 我们计划研究两种DNA解旋酶。结果表明，E. Coli.RecBCD酶是一种多亚基、多功能酶， 同时具有解旋酶和核酸酶活性；它还具有 为了在移位时识别被称为khi的特定DNA序列， 这调节了它的核溶解活动。希望能有一个详细的 这些解旋酶的生化比较将有助于阐明 DNA解旋酶的生化机制和生物学功能。 这项提案的长期、具体目标是继续我们的 沙门氏菌DNA解旋酶和核酸酶活性的研究 RecBCD酶，以及这些活性是如何 受重组热点序列KHi相互作用的影响。 此外，我们希望将这些发现与 RecQ蛋白。 这些知识将有助于对分子的一般理解。 导致细胞异常过程的事件。基因突变 编码可能的解旋酶的基因与人类疾病有关，如 多种多样的色素性干皮病(ERCC2和ERCC3)；Cockayne综合征 (EERCC6)；Bloom综合征(BLM，它是RecQ同源物)；以及 Werner综合征(WRN，也是RecQ同源基因)，表明 对这些蛋白质的理解对于理解 疾病的过程多种多样，如癌症和过早衰老。

DNA helicases represent a ubiquitous class of nucleic acid enzymes. These proteins "unwind" (i.e., denature) dsDNA into separate single- strands of DNA, in a reaction requiring nucleoside triphosphate hydrolysis. The broad objective of this proposal is to understand the biochemical mechanism and biological function of DNA helicase action, i.e., how helicases translocate along single-stranded DNA (ssDNA) to unwind double-stranded (dsdna) and, having done so, how this effort is translated into biological function (i.e., useful work). An understanding of these fundamental questions will contribute to our understanding of the function of these helicases and of translocating proteins, in general. We plan to study two DNA helicases. The RecBCD and RecQ enzymes of E. coli. The RecBCD enzyme is a multi-subunit, multifunctional enzyme that possesses both helicase and nuclease activities; it also has the ability to recognize, while translocating, a specific DNA sequence called khi, that regulates its nucleolytic activities. It is hoped that a detailed biochemical comparison of these helicases will help clarify the biochemical mechanism and biological function of DNA helicases. The long-term, specific aims of this proposal are to continue our investigations into the DNA helicase and nuclease activities of the RecBCD enzyme, and into the manner by which these activities are affected by interaction with the recombination hotspot sequence, khi. Furthermore, we wish to compare these findings to the behavior of the RecQ protein. This knowledge will contribute a general appreciation of the molecular events responsible for aberrant cellular processes. Mutations in genes that encode putative helicases are associated with human disorders as diverse as xeroderma pigmentosum (ERCC2 and ERCC3); Cockayne's syndrome (EERCC6); Bloom's syndrome (BLM, which is a RecQ homologue); and Werner's syndrome (WRN, which is also a RecQ homologue), showing that an understanding of these proteins is crucial for the understanding of disease processes as diverse as cancer and premature aging.