BIOCHEMICAL MECHANISM AND FUNCTION OF DNA HELICASES

DNA解旋酶是一类普遍存在的核酸酶。 这些蛋白质“解旋”（即，变性）dsDNA分离成单独的单- 在需要三磷酸核苷的反应中， 水解 本提案的总体目标是了解 DNA解旋酶作用的生化机制和生物学功能， 也就是说，解旋酶如何沿着沿着单链DNA（ssDNA）移位， 解开双链（dsdna），并在这样做之后，这项工作是如何进行的？ 转化为生物学功能（即，有用的工作）。 一个 了解这些基本问题将有助于我们 了解这些解旋酶的功能和易位 蛋白质，总的来说。 我们计划研究两种DNA解旋酶。 E. 杆菌 RecBCD酶是一种多亚基、多功能酶， 具有解旋酶和核酸酶活性;它还具有 来识别一种叫做khi的特定DNA序列， 调节其溶核活性。 希望能有一个详细的 这些解旋酶的生物化学比较将有助于阐明 DNA解旋酶的生物学功能。 这项提案的长期具体目标是继续我们的 研究了DNA解旋酶和核酸酶活性， RecBCD酶，并进入这些活动的方式， 受与重组热点序列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生物学的一般认识。 导致异常细胞过程的事件。 基因突变 编码推定解旋酶的基因与人类疾病有关， 多种多样，如着色性干皮病（ERCC 2和ERCC 3）; Cockayne综合征 Bloom综合征（BLM，其为RecQ同源物）;和 沃纳综合征（WRN，也是RecQ同源物），表明 对这些蛋白质的了解对于理解 癌症和过早衰老等多种疾病。

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.