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

DNA 解旋酶在维持基因组稳定性所需途径中的作用

• 批准号: 8148305
• 负责人: Robert Brosh
• 金额: $ 23.45万
• 依托单位: NATIONAL INSTITUTE ON AGING
• 依托单位国家: 美国
• 资助国家: 美国
• 起止时间: 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/8148305
• 关键词: Genome Stability; Maintenance; Pathway interactions; Role; helicase

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.

解旋酶是分子运动蛋白，将三磷酸核苷的水解与核酸脱落。该类别的酶与其他蛋白质作为复杂的机器协同功能，并在DNA代谢途径中起重要作用，包括复制，DNA修复，重组，转录和染色体分离。尽管为了解解旋酶功能的生化，结构和遗传方面的努力做出了巨大努力，但旋转酶催化链分离并履行其生物学作用的确切机制仍有待完全理解。 与人类疾病有关的DNA解旋酶的数量越来越多，表明这些酶在细胞途径中对维持基因组稳定性很重要。 最近的证据表明，DNA解旋酶的RECQ家族的突变导致过早衰老和/或癌症易感性的染色体不稳定性疾病。目前已知的RECQ解旋酶缺陷疾病包括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/核酸酶，大概在DNA代谢中起作用以保持基因组完整性。为了了解WRN影响的DNA结构和细胞途径，我们系统地检查了WRN解旋酶的DNA底物偏好，以便放松及其与人类核蛋白的相互作用。我们的生化研究表明，WRN优先放松定义方向的DNA复制结构，并利用特定的DNA结构元素进行识别。我们的小组使用了对WRN解旋酶活性的实时动力学分析，以表征WRN解除DNA的机制。 进行了生化研究，以研究其辅助因子RPA刺激WRN解旋酶活性的机制。 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关键作用。为了进一步了解WRN蛋白的分子功能，我们表征了WRN与人瓣内切核酸酶1（FEN-1）的功能相互作用，这是一种与DNA修复，复制和重组有关的结构特异性核酸酶。我们的结果表明，WRN通过独特的机制刺激重要DNA中间体的Fen-1裂解，从而大大提高了Fen-1裂解的效率。我们最近的工作阐明了WRN在解决停滞的复制叉和重组中间体中的作用。我们的假设是，异常的有丝分裂重组和基因组不稳定性是由于对Werner综合征细胞中复制/重组中间体的不适当处理而产生的。 使用模型遗传系统用于WRN结构功能研究，可以在体内证据表明WRN在细胞DNA复制中的作用。 尽管已广泛研究了WRN和BLM解旋酶的生化特性和蛋白质相互作用，但有关其他人RECQ解旋酶功能的信息较少。我们将注意力集中在人类RECQ1上，它是一种DNA解旋酶，其细胞功能在很大程度上没有表征。发现RECQ1稳定地结合了各种DNA结构，从而使其能够放松一组不同的DNA底物。显示RECQ1可在互补的单链DNA分子之间催化有效的链退火。为了更好地了解RECQ1细胞功能，我们研究了其蛋白质相互作用。我们的结果表明，REC1通过与不匹配修复因子的相互作用来调节遗传重组的作用。 当前，我们正在利用模型系统来确定RECQ1的生物学功能。