ACETYLATION OF HUMAN HELICASE/NUCLEASE

这个子项目是许多研究子项目中利用 资源由NIH/NCRR资助的中心拨款提供。子项目和 调查员(PI)可能从NIH的另一个来源获得了主要资金， 并因此可以在其他清晰的条目中表示。列出的机构是 该中心不一定是调查人员的机构。 RecQ解旋酶基因BLm编码人类细胞中的五种RecQ解旋酶之一。编码其中三种解旋酶WRN、RECQ4和BLM的基因突变会导致以过早衰老和/或癌症易感性为特征的疾病。BLM的纯合子突变会导致被称为布卢姆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。布鲁姆患者几乎容易患上所有类型的癌症。由于复制分叉的基本过程的复杂性，以及我们对真核生物复制机制的有限了解，尽管多年来对BLM保护基因组的具体机制的了解是不完整的，但对BLM保护基因组的具体机制的了解仍然不完整。在这一机制中，额外的DNA解旋酶执行基本功能。我们发现人BLM肿瘤抑制基因在酵母中的功能是抑制另一种解旋酶DNA2解旋酶缺陷的突变株的DNA复制和DNA修复缺陷。我们建议利用这一观察来理解人类BLM以及它是如何保持基因组完整性的。DNA2参与冈崎片段的处理，这是维持高保真DNA复制的关键步骤。因此，对DNA2突变体的抑制首次表明BLM在Okazaki片段处理(OFP)中发挥关键作用。我们认为，由于复制体和修复体的复杂性，解决人BLM抑制DNA2的相关性的唯一确定的方法是使用一个定义的复制/修复系统，该系统完全由纯化的蛋白质和定义的模仿OFP中间体的寡核苷酸底物组成。我们将利用冈崎片段加工的不同阶段的生化重组来确定BLM解旋酶的具体作用。为了利用在单细胞真核生物酵母中进行复制和重组的更简单的环境，到目前为止，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。我们将直接研究复制的DNA2解旋酶与Bloom在人类细胞中的相互作用。我们将对人类DNA2的定位进行细胞生物学分析，重点是端粒，因为在酵母中DNA2是动态定位于端粒的。

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.