ACETYLATION OF HUMAN HELICASE/NUCLEASE

这个子项目是许多研究子项目中的一个 由NIH/NCRR资助的中心赠款提供的资源。子项目和 研究者（PI）可能从另一个NIH来源获得主要资金， 因此可以在其他CRISP条目中表示。所列机构为 研究中心，而研究中心不一定是研究者所在的机构。 RecQ解旋酶基因BLM编码人类细胞中的五种RecQ解旋酶之一。 编码这些解旋酶中的三种WRN、RECQ 4和BLM的基因的突变引起以过早衰老和/或癌症易感性为特征的疾病。 BLM中的纯合子突变导致称为Bloom综合征的癌症易感性障碍。 因此，BLM被归类为看守肿瘤抑制因子。 布卢姆病患者几乎容易患上所有类型的癌症。 知识的具体机制，BLM保护基因组，尽管多年的一般表征，是不完整的，由于在复制叉的基本过程的复杂性和我们有限的理解在真核生物中的复制机制。在这种机制中，额外的DNA解旋酶执行基本功能。 我们已经发现，人BLM肿瘤抑制基因在酵母中的功能是抑制另一种解旋酶Dna 2解旋酶缺陷突变体中的DNA复制和DNA修复缺陷。 我们建议利用这一观察来了解人类BLM及其如何保持基因组完整性。 DNA 2参与冈崎片段加工，这是维持高保真DNA复制的关键步骤。 因此，抑制dna 2突变体首次表明，BLM在冈崎片段加工（OFP）中起着至关重要的作用。 我们相信，由于复制体和修复体的复杂性，解决人BLM抑制dna 2的相关性的唯一确定的方法是使用完全由纯化的蛋白质和模拟OFP中间体的确定的寡核苷酸底物组成的确定的复制/修复系统。 我们将使用冈崎片段加工的各个阶段的生化重建来确定BLM解旋酶的具体作用。 为了利用在单细胞真核生物酵母中发生复制和重组的更简单的环境，迄今为止我们的研究仅限于酵母模型系统，但现在将开始将我们的研究扩展到人类细胞。 我们将直接研究人类细胞中复制型DNA 2解旋酶和Bloom之间的相互作用。 我们将进行细胞生物学分析的本地化的人类DNA 2，集中在端粒，因为在酵母DNA 2是动态本地化的端粒。

This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. The RecQ helicase gene, BLM, encodes one of five RecQ helicases in human cells. Mutations in the genes encoding three of these helicases, WRN, RECQ4, and BLM, cause diseases characterized by premature aging and/or cancer predisposition. Homozygous mutations in BLM lead to the cancer predisposition disorder known as Bloom syndrome. Thus, BLM is classified as a caretaker tumor suppressor. Bloom patients are prone to almost all classes of cancer. Knowledge of the specific mechanisms by which BLM protects the genome, despite years of general characterization, is incomplete due to the complexity of the underlying processes at the replication fork and our limited understanding of the replication machinery in eukaryotes. In this machinery, additional DNA helicases perform essential functions. We have found that the human BLM tumor suppressor gene functions in yeast to suppress both the DNA replication and DNA repair defects in mutants defective in another helicase, Dna2 helicase. We propose to use this observation to understand human BLM and how it preserves genome integrity. Dna2 is involved in Okazaki fragment processing, a critical step for maintaining high fidelity DNA replication. Suppression of dna2 mutants therefore suggests for the first time that BLM plays a critical role in Okazaki fragment processing (OFP). We believe that due to the complexity of replisomes and repairosomes, the only sure way to address the relevance of the suppression of dna2 by human BLM is to use a defined replication/repair system consisting entirely of purified proteins and defined oligonucleotide substrates that mimic intermediates in OFP. We will use biochemical reconstitution of the various stages of Okazaki fragment processing to identify the specific role of the BLM helicase. To take advantage of the simpler milieu in which replication and recombination take place in the unicellular eukaryote, yeast, we have limited our studies to date to the yeast model system, but will now begin to extend our studies to human cells. We will investigate the interaction between the replicative Dna2 helicase and Bloom in human cells directly. We will carry out cell biological analysis of the localization of human Dna2, focusing on telomeres, since in yeast Dna2 is dynamically localized to telomeres.