DNA helicases and associated factors in genome stability

项目摘要 DNA解旋酶代表了在DNA复制的几乎所有方面起作用的极少数酶类别之一， 重组、修复和端粒维持。因此，它们对维持基因组完整性至关重要， 突变后会导致疾病因此，迫切需要全面了解解旋酶生物学 以及这些酶如何支持基因组的完整性。尽管在体内和体外研究中取得了许多进展， 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方面存在知识空白 在诊所里。本研究的目的是从分子机理上深入了解DNA解旋酶在基因组中的作用 以及为什么它们的功能障碍会导致疾病。为了实现这一目标，我们正在研究PIF 1和RecQ 家族解旋酶，因为它们在生命的所有领域都是进化保守的（给了我们选择模型的机会 系统来剖析其生物学的各个方面），并且因为人PIF 1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 以及易患癌症和过早衰老。我们目前的工作集中在RecQ解旋酶的作用 在DNA链间交联（ICL）修复以及RecQ和Pif 1解旋酶中端粒维持。拟议 工作将：1）确定RecQ 4亚家族解旋酶和Pso 2家族核酸酶在范可尼贫血中的功能- 独立的DNA ICL修复，确定参与这一修复途径的其他因素，并研究疾病 RECQL 4的等位基因在机制水平上干扰其基因组维持功能; 2）定义PIF 1和 RecQ解旋酶协同调节端粒酶活性，确定其他端粒结合的影响 这些解旋酶的生物化学上的蛋白质，并在体外重建端粒酶全酶;和3） 确定赖氨酸乙酰化对酵母和人类中PIF 1家族解旋酶的调节作用，以及这种调节作用是如何产生的。 与基因组的完整性有关为了完成这项工作，我们将聘请各种经典和尖端的 实验技术，从标准的体外酶测定和模式生物遗传学到下一个- 代测序、交联质谱和定制点击化学探针的开发。 总的来说，这项工作将提供关键的基础数据，了解PIF 1和RecQ家族解旋酶如何 有助于维持基因组的稳定性，并最终导致治疗目标和治疗方法， 解旋酶相关疾病。

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.