Function of the Bloom's syndrome DNA helicase in the maintainance of genome integrity

布卢姆氏综合征 DNA 解旋酶在维持基因组完整性中的功能

• 批准号: 10254408
• 负责人: Kristina Schmidt
• 金额: $ 33.78万
• 依托单位: UNIVERSITY OF SOUTH FLORIDA
• 依托单位国家: 美国
• 财政年份: 2020
• 资助国家: 美国
• 起止时间: 2020-09-04 至 2024-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10254408
• 关键词: ATP phosphohydrolase; Acceleration; Active Sites; Base Pairing; Binding; Binding Sites; Bloom Syndrome; Bloom syndrome protein; Cell Cycle; Cell physiology; Cells; Chromatin; Chromosomal Instability; Chromosomal Stability; Chromosome Breakage; Chromosomes; Complex; Coupling; Cruciform DNA; Crystallography; DNA; DNA Binding; DNA Damage; DNA Double Strand Break; DNA Repair; DNA Structure; DNA biosynthesis; DNA replication fork; Data; Defect; Development; Disease; Double Strand Break Repair; Enzymes; Eukaryotic Cell; Excision; G-Quartets; G1 Phase; G1/S Transition; GTP-Binding Protein alpha Subunits, Gs; Genome; Genome Stability; Genomic Instability; Health; Human; Human Genome; Licensing Factor; Life Expectancy; Maintenance; Malignant Neoplasms; Mammalian Cell; Mitosis; Mitotic; Modeling; Molecular; Mutation; N-terminal; Play; Predisposition; Proteome; Recovery; Regulation; Replication Initiation; Replication Origin; Resolution; Role; S Phase; Site; Speed; Structure; Syndrome; Tail; Testing; base; biophysical techniques; cancer cell; cancer predisposition; cancer risk; cancer type; design; genome integrity; helicase; homologous recombination; improved; in vivo; insight; molecular dynamics; mutant; novel; prevent; recruit; response; single-molecule FRET

PROJECT SUMMARY The RecQ-like DNA helicase BLM is known for its critical role in the response to and repair of DNA-double- strand breaks in mammalian cells. Disruption of BLM activity causes Bloom’s syndrome, which is characterized by extreme cancer risk, short stature, and an average life expectancy of 25 years. Cancer susceptibility, chromosome breakage and other cellular defects are currently explained by the lack of BLM’s activity in the DNA-damage response and homologous recombination. In this proposal we are testing the hypothesis that BLM plays critical roles in DNA replication initiation and elongation to maintain chromosome stability in unperturbed cells. This hypothesis is based on extensive preliminary data, including an unbiased screen of the mid-S-phase proteome that led to the discovery that chromatin-bound BLM directly interacts with the Mcm6 subunit of chromatin-bound Mcm2-7. Notably, two distinct binding sites in BLM and Mcm6 differentially regulate complex formation in G1 and S-phase, and disruption of the BLM/Mcm6 interaction in S-phase, but not in G1, leads to supra-normal DNA replication speed. Aberrant acceleration of DNA replication speed beyond a safe limit is emerging as a mechanism that causes DNA damage and kills certain types of cancer cells. Our preliminary findings suggest that the BLM/Mcm6 interaction acts as a novel, negative regulator of DNA replication in human cells. That cells lacking BLM do not exhibit increased replication speed suggests that acceleration of replication requires the BLM protein, leading us to hypothesize that BLM needs to be tethered to Mcm6 to restrict the ATPase/helicase activity of BLM to the immediate vicinity of the replisome. Together with BLM’s ability to unwind G-quadruplexes (G4s) and their presence throughout the human genome, including at ~90% of origins of replication, we propose that BLM is recruited by Mcm6 to unfold DNA structures (i) at replication origins to facilitate the G1/S transition (Aim 1) and (ii) throughout the genome to regulate replisome progression during unperturbed S-phase (Aim 2). We have isolated a set of BLM mutants that specifically fail to interact with Mcm6 in G1 or S-phase, or both, to identify the separate functions of the BLM/Mcm6 interaction in G1 and S-phase and to determine replication-associated mitotic defects. Further, we will use biophysical approaches and molecular dynamics simulations to determine the mechanism of G4 unwinding by BLM (Aim 3). Completing these studies will delineate a major new function for BLM in unperturbed DNA replication, besides its established role in DNA double-strand break repair and replication fork restart after DNA damage, and determine its mechanism of G4 unwinding. Our findings will provide a major advance in our understanding of the mechanisms that prevent chromosome instability in unperturbed cells and improve our understanding of chromosome breakage syndromes and cancer predisposition.

项目总结 RECQ样DNA解旋酶BLm是已知的在DNA-Double-DNA反应和修复中的关键角色. 哺乳动物细胞中的链断裂。BLM活动中断会导致Bloom综合征，其特征是 癌症风险极高，身材矮小，平均预期寿命为25岁。癌症易感性， 目前解释染色体断裂和其他细胞缺陷的原因是BLM在 DNA损伤反应和同源重组。在这个提案中，我们正在检验这样一个假设： BLm在DNA复制的起始和延伸以维持染色体稳定性方面起着关键作用 在不受干扰的牢房里。这一假设是基于广泛的初步数据，包括对 发现染色质结合的BLm直接与Mcm6相互作用的S中期蛋白质组 染色质结合的Mcm2-7亚基。值得注意的是，BLM和Mcm6上的两个不同的结合位点是不同的 在G1期和S期调节复合体的形成，在S期破坏BLm/Mcm6相互作用，但 不是在G1中，导致超乎正常的DNA复制速度。DNA复制速度异常加快 超过安全限度正在成为一种导致DNA损伤和杀死某些类型癌症的机制 细胞。我们的初步发现表明，BLM/Mcm6相互作用是一种新的负性调节因子 人类细胞中的DNA复制。缺乏博莱姆的细胞不会表现出更快的复制速度 提示复制的加速需要BLM蛋白，这让我们假设BLM需要 将其与Mcm6连接，将BLm的ATPase/解旋酶活性限制在其附近 复制体。再加上BLM解除G-四联体(G4S)的能力以及它们在整个 人类基因组，包括在~90%的复制起点，我们认为BLM是由Mcm6招募的 在复制起点展开dna结构(I)以促进G1/S的过渡(目标1)和(Ii)在整个 基因组在无动于衷的S期调控复制体进程(目标2)。我们已经分离出一组 在G1期或S期或两者都未能与Mcm6相互作用的BLm突变体，以识别单独的 BLm/Mcm6相互作用在G1期和S期的作用及其对复制相关有丝分裂的影响 缺陷。此外，我们将使用生物物理方法和分子动力学模拟来确定 博莱曼对G4解链的机制(目标3)。完成这些研究将勾勒出一个主要的新功能 BLM除了在DNA双链断裂修复和修复中的既定作用外，还在未受干扰的DNA复制中发挥作用 复制叉在DNA损伤后重新启动，并确定其G4解离机制。我们的发现将 为我们理解防止染色体不稳定的机制提供了重大进展 不受干扰的细胞，并提高我们对染色体断裂综合征和癌症的理解 性情。