DNA helicases and associated factors in genome stability

项目总结 DNA解旋酶是极少数在DNA复制的几乎所有方面起作用的酶类之一， 重组、修复和端粒维持。因此，它们对于维持基因组的完整性和 当突变时，疾病会联系在一起。因此，有必要全面了解解旋酶生物学。 以及这些酶是如何支持基因组完整性的。尽管在体内和体外研究方面取得了许多进展 解旋酶，在将解旋酶基因的突变等位基因与患者的治疗联系起来的知识方面存在差距 在诊所里。我研究的目的是从机制上了解dna解旋酶在基因组中的功能。 以及为什么它们的功能障碍会导致疾病。为了达到这个目标，我们正在研究PIF1和RECQ 家族解旋酶，两者都是因为它们在进化上在生命的所有领域都是保守的(给了我们选择模型 系统来剖析它们生物学的各个方面)，因为人类PIF1和RecQ解旋酶是 致癌基因。事实上，编码这些解旋酶的基因突变与多种疾病有关，如 以及癌症和过早衰老的易感性。我们目前的工作集中在RecQ解旋酶的作用上 在DNA链间交联(ICL)修复和端粒维持中的RecQ和Pif1解旋酶。建议数 工作将：1)确定RecQ4亚家族解旋酶和Pso2家族核酸酶在Fanconi贫血中的作用- 独立的DNA ICL修复，确定参与该修复途径的其他因素，并检查疾病 RECQL4的等位基因在机制水平上扰乱了它的基因组维持功能；2)定义了PIF1和 RecQ解旋酶协同调节端粒酶活性，决定其他端粒结合的影响 蛋白质对这些解旋酶的生化作用，并在体外重建端粒酶全酶；以及3) 确定赖氨酸乙酰化对酵母和人类中PIF1家族解旋酶的调节作用以及这是如何实现的 与基因组的完整性有关。为了完成这项工作，我们将采用各种经典和尖端的 实验技术，从标准的体外酶分析和模型生物遗传学到下一步- 世代测序、交联质谱，以及定制点击化学探针的开发。 总体而言，这项工作将为理解PIF1和RecQ家族解旋酶提供关键的基础数据 有助于维持基因组的稳定性，并最终将导致治疗目标和治疗 与解旋酶相关的疾病。

PROJECT SUMMARY DNA helicases represent one of very few enzyme classes that function in virtually all aspects of DNA replication, recombination, repair, and telomere maintenance. As such, they are vital to maintaining genome integrity and are disease linked when mutated. Thus, there is a critical need to comprehensively understand helicase biology and how these enzymes support genome integrity. Despite many in vivo and in vitro advances in working with helicases, there is a gap in knowledge connecting mutant alleles of helicase genes to the treatment of patients in clinics. The objective of my research is to gain mechanistic insight into how DNA helicases function in genome maintenance and why their dysfunction leads to disease. Toward this goal, we are studying PIF1 and RecQ family helicases, both because they are evolutionarily conserved in all domains of life (giving us our pick of model systems to dissect the various aspects of their biology) and because the human PIF1 and RecQ helicases are oncogenes. Indeed, mutations in the genes encoding these helicases are associated with multiple diseases, as well as predispositions to cancers and premature aging. Our current work focuses on the roles of RecQ helicases in DNA inter-strand crosslink (ICL) repair and RecQ and Pif1 helicases in telomere maintenance. The proposed work will: 1) determine how RecQ4 subfamily helicases and Pso2 family nucleases function in Fanconi anemia- independent DNA ICL repair, identify other factors involved in this repair pathway, and examine how disease alleles of RECQL4 perturb its genome maintenance functions at a mechanistic level; 2) define how PIF1 and RecQ helicases synergistically modulate telomerase activity, determine the impacts of other telomere binding proteins on the biochemistry of these helicases, and reconstitute the telomerase holoenzyme in vitro; and 3) determine the regulatory effects of lysine acetylation on PIF1 family helicases in yeast and humans and how this is linked to genome integrity. To perform this work, we will employ a variety of classic and cutting edge experimental techniques, from standard in vitro enzymatic assays and model organism genetics to next- generation sequencing, crosslinking mass spectrometry, and the development of custom click chemistry probes. Overall, this work will provide fundamental data critical to understanding how PIF1 and RecQ family helicases aid in the maintenance of genome stability, and it will ultimately lead to therapeutic targets and treatments for helicase-linked diseases.