Roles Of DNA Helicases In Pathways Required For Maintenance Of Genomic Stability

解旋酶是一种分子马达蛋白，它将核苷三磷酸的水解与核酸解离结合起来。这类酶作为一个复杂的机器与其他蛋白质协调工作，在DNA代谢途径中发挥重要作用，包括复制、DNA修复、重组、转录和染色体分离。尽管人们在了解解旋酶功能的生化、结构和遗传方面做了大量的工作，但解旋酶催化链分离和发挥其生物学作用的确切机制仍不完全清楚。越来越多的DNA解旋酶与人类疾病有关，这表明这些酶在细胞通路中具有重要的专门作用，对维持基因组的稳定至关重要。 最近的证据表明，RECQ家族DNA解旋酶基因的突变会导致染色体不稳定，导致过早衰老和/或癌症易感性。目前已知的RecQ解旋酶缺陷性疾病包括Werner、Bloom和Rothmund-Thomson综合征。WRN基因产物在Werner综合征中有缺陷，是一种解旋酶/核酸外切酶，可能在DNA新陈代谢中发挥作用，以保持基因组的完整性。为了了解WRN影响的DNA结构和细胞途径，我们系统地研究了WRN解旋酶的DNA底物偏好及其与人类核蛋白的相互作用。我们的生化研究表明，WRN优先在定义的方向上解开DNA复制结构，并利用特定的DNA结构元件进行识别。我们小组使用WRN解旋酶活性的实时动力学分析来表征WRN解开DNA的机制。对其辅助因子RPA刺激WRN解旋酶活性的机制进行了生化研究。我们的结果表明，RPA和WRN之间的物理相互作用在功能相互作用中起着关键作用。为了进一步了解WRN蛋白的分子功能，我们研究了WRN与人翻盖核酸酶1(FEN-1)的功能相互作用，FEN-1是一种参与DNA修复、复制和重组的结构特异性核酸酶。我们的结果表明，WRN通过一种独特的机制刺激重要DNA中间体的FEN-1裂解，从而显著提高FEN-1的裂解效率。我们最新的工作阐明了WRN在解决复制分叉和重组中间体停滞中的作用。我们的假设是，Werner综合征细胞的异常有丝分裂重组和基因组不稳定源于复制/重组中间产物的不适当处理。使用WRN结构-功能研究的模型遗传系统，在体内获得了WRN在细胞DNA复制中的作用的证据。 虽然WRN和BLM解旋酶的生化特性和蛋白质相互作用已被广泛研究，但关于其他人类RecQ解旋酶的功能的信息较少。我们将注意力集中在人类RECQ1上，这是一种DNA解旋酶，其细胞功能在很大程度上仍未确定。RECQ1被发现能够稳定地结合各种DNA结构，使其能够解开一组不同的DNA底物。结果表明，RECQ1能有效地催化互补单链DNA分子间的链退火。为了更好地了解RECQ1的细胞功能，我们研究了它的蛋白质相互作用。我们的结果表明，RECQ1通过与错配修复因子的相互作用，在基因重组的调控中发挥作用。目前，我们正在利用模型系统来确定RECQ1的生物学功能。

Helicases are molecular motor proteins that couple the hydrolysis of nucleoside triphosphate to nucleic acid unwinding. Enzymes of this class function coordinately with other proteins as a complex machine and play essential roles in pathways of DNA metabolism that include replication, DNA repair, recombination, transcription, and chromosome segregation. Despite considerable efforts to understand biochemical, structural, and genetic aspects of helicase function, the precise mechanisms by which helicases catalyze strand separation and perform their biological roles remain to be fully understood. The growing number of DNA helicases implicated in human disease suggests that these enzymes have vital specialized roles in cellular pathways important for the maintenance of genome stability. Recent evidence indicates that mutations in genes of the RecQ family of DNA helicases result in chromosomal instability diseases of premature aging and/or cancer predisposition. Currently known RecQ helicase-deficient disorders include Werner, Bloom, and Rothmund-Thomson syndromes. The WRN gene product, defective in Werner syndrome, is a helicase/exonuclease that presumably functions in DNA metabolism to preserve genome integrity. To understand the DNA structures and cellular pathways that WRN impacts, we have systematically examined the DNA substrate preferences of WRN helicase for unwinding and its interactions with human nuclear proteins. Our biochemical studies indicate that WRN preferentially unwinds DNA replication structures in a defined orientation and utilizes specific DNA structural elements for recognition. A real-time kinetic analysis of WRN helicase activity was used by our group to characterize the mechanism of DNA unwinding by WRN. Biochemical studies were performed to investigate the mechanism for stimulation of WRN helicase activity by its auxiliary factor RPA. Our results indicate that the physical interaction between RPA and WRN plays a critical role in the functional interaction. To further understand the molecular functions of WRN protein, we have characterized the functional interaction of WRN with human Flap Endonuclease 1 (FEN-1), a structure-specific nuclease implicated in DNA repair, replication, and recombination. Our results indicate that WRN stimulates FEN-1 cleavage of important DNA intermediates by a unique mechanism whereby the efficiency of FEN-1 cleavage is dramatically enhanced. Our most recent work has elucidated a role for WRN in resolving stalled replication forks and recombination intermediates. Our hypothesis is that the aberrant mitotic recombination and genomic instability arises from inappropriate processing of replication/recombination intermediates in Werner syndrome cells. In vivo evidence for a role of WRN in cellular DNA replication was attained using a model genetic system for WRN structure-function studies. Although the biochemical properties and protein interactions of the WRN and BLM helicases have been extensively investigated, less information is available concerning the functions of the other human RecQ helicases. We have focused our attention on human RECQ1, a DNA helicase whose cellular functions remain largely uncharacterized. RECQ1 was found to stably bind a variety of DNA structures, enabling it to unwind a diverse set of DNA substrates. RECQ1 was shown to catalyze efficient strand annealing between complementary single-stranded DNA molecules. To acquire a better understanding of RECQ1 cellular functions, we have investigated its protein interactions. Our results suggest a role of RECQ1 in regulation of genetic recombination by its interaction with mismatch repair factors. Currently, we are utilizing model systems to determine the biological functions of RECQ1.