Telomere maintenance and replication fork progression in yeast and human cells

 描述（由申请人提供）：Zakian实验室的长期研究目标是促进对促进真核染色体完整性机制的理解。拟议的工作分为两个领域福尔斯。第一个领域是端粒的维护，染色体的末端，通过端粒酶，使用芽殖和分裂酵母作为模型。第二个领域是复制叉通过难以复制的位点的进展以及Pif 1家族DNA解旋酶在这一过程中的作用。建议的复制实验将在芽殖酵母和人类培养细胞中进行。这两个研究领域通过Pif 1家族解旋酶的作用联系在一起：芽殖酵母Pif 1是端粒酶的负调节剂，而其他Pif 1家族解旋酶促进端粒的半保守DNA复制，以及其他难以复制的位点。DNA的两种结构变体也将这两个区域联系起来。G-四链体（G4）结构，其可以在RNA或单链DNA中形成适当的序列，通过非典型的G-G碱基对保持在一起。R环是通常在异常转录期间形成的RNA-DNA杂合体。G4 DNA和r环都是稳定的，都是缓慢的复制叉并导致DNA损伤，都在端粒和其他难以复制的位点形成。这两个区域之间的另一个联系是芽殖酵母Pif 1解旋酶非常有效地解绕G4 DNA和r环。端粒酶调控的实验将利用我们成功地使用质谱（MS），以确定新的芽殖酵母端粒酶调节剂与端粒酶全酶。该方法将Cdc 48-Npl 4-Ufd 1复合物鉴定为Est 1丰度和活性的调节剂。我们还鉴定了RNAseP/MRP的多个亚基与端粒酶相关，并将继续探索这种关联如何影响端粒酶RNA。在下一个资助期内，我们将把MS方法扩展到裂变酵母端粒酶，我们预计这将同样富有成效。我们还将使用MS来识别芽殖酵母中关键端粒蛋白的翻译后修饰和蛋白质-蛋白质相互作用，作为端粒长度的函数，使用两种遗传策略来生成所有细胞都具有短或长端粒的培养物。我们正在进行芽殖酵母Pif 1的结构-功能分析的功能部分，目的是确定底物特异性残基，使我们能够将ScPif 1的不同作用与其独特的生化活性联系起来。我们正在使用重组腺相关病毒（rAAV）和CRISPR/Cas9技术的组合来构建缺乏人PIF 1的人类组织培养系，目的是确定其DNA靶点以及其突变如何与家族性乳腺癌相关。分析将包括将靶序列克隆到载体中， 通过二维凝胶监测哺乳动物细胞中的复制。我们正在使用一种新的方法来确定G4结构和r-环在芽殖酵母全基因组和核糖体DNA在不同的突变体和生长条件下的网站，其目标是提供物理证据的结构和遗传证据，它们的相互作用在体内的存在。

 DESCRIPTION (provided by applicant): The long-term research goal of the Zakian lab is to contribute to the understanding of mechanisms that promote the integrity of eukaryotic chromosomes. Proposed work falls into two areas. The first area is maintenance of telomeres, the ends of chromosomes, by telomerase, using budding and fission yeast as models. The second area is the progression of replication forks through hard-to-replicate sites and the roles of Pif1 family DNA helicases in this process. The proposed replication experiments will be carried out in budding yeast and human cultured cells. The two areas of research are linked by the role of Pif1 family helicases: budding yeast Pif1 is a negative regulator of telomerase while other Pif1 family helicases promote semi- conservative DNA replication of telomeres, as well as of other hard-to-replicate sites. There are two structural variants of DNA that also link the two areas. G-quadruplex (G4) structures, which can form in RNA or single- stranded DNA of the appropriate sequence, are held together by non-canonical G-G base pairs. R-loops are RNA-DNA hybrids that usually form during aberrant transcription. G4 DNA and r-loops are both stable, both slow replication forks and cause DNA damage, and both form at telomeres and other hard to replicate sites. Another link between the two areas is that the budding yeast Pif1 helicase unwinds G4 DNA and r-loops very efficiently. Experiments on regulation of telomerase will exploit our success in using mass spectrometry (MS) to identify novel regulators of budding yeast telomerase by their association with the telomerase holoenzyme. This approach identified the Cdc48-Npl4-Ufd1 complex as a regulator of Est1 abundance and activity. We also identified multiple subunits of RNAseP/MRP as being telomerase associated and will continue to explore how this association affects telomerase RNA. In the next funding period, we will extend the MS approach to fission yeast telomerase, which we anticipate will be equally fruitful. We will also use MS to identify post-translational modifications and protein-protein interactions of key telomere proteins in budding yeast as a function of telomere length using two genetic strategies to generate cultures where all cells have short or long telomeres. We are carrying out the function part of a structure-function analysis of budding yeast Pif1 with the goal of identifying substrate-specific residues that will enable us to connect the disparate roles of ScPif1 to its distinct biochemical activities. We are using a combination of recombinant adeno-associated viruses (rAAV) and CRISPR/Cas9 technology to construct human tissue culture lines that lack human PIF1 with the goal of determining its DNA targets and how its mutation is associated with familial breast cancer. Analysis will include cloning target sequences into a vector that allows us to monitor replication in mammalian cells by two- dimensional gels. We are using a novel approach to identify sites of G4 structures and r-loops in budding yeast genome-wide and in the ribosomal DNA in different mutants and growth conditions with the goal of providing physical evidence for the in vivo existence of both structures and genetic evidence for their interaction.