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.

项目摘要 RECQ样DNA解旋酶BLM以其在对DNA双 - 双重响应和修复中的关键作用而闻名 链条中断哺乳动物细胞。 BLM活动的破坏会导致Bloom的综合征，这是特征的 由于极端的癌症风险，短期统计和平均预期寿命为25年。癌症的敏感性， 染色体断裂和其他细胞缺陷目前的解释是由于BLM缺乏活性 DNA破坏反应和同源重组。在此提案中，我们正在检验以下假设 BLM在DNA复制计划和伸长率中扮演着关键的作用，以保持染色体稳定性 在不受干扰的细胞中。该假设基于广泛的初步数据，包括 导致发现染色质BLM直接与MCM6相互作用的发现中的S相蛋白质组 结合染色质MCM2-7的亚基。值得注意的是，BLM和MCM6中的两个不同的结合位点差异化 调节G1和S期间的复合物形成，并破坏S相间的BLM/MCM6相互作用，但 不在G1中，导致超正常的DNA复制速度。 DNA复制速度的异常加速度 超出安全限制是作为造成DNA损害并杀死某些类型癌症的机制。 细胞。我们的初步发现表明，BLM/MCM6相互作用是一种新颖的负调节剂 人类细胞中的DNA复制。缺乏BLM的细胞不存在增加复制速度 表明复制的加速需要BLM蛋白，使我们假设BLM需求 要束缚在MCM6上以限制BLM的ATPase/Helicase活性到附近 复制体。加上BLM的放松G-四链体（G4S）及其在整个过程中的能力 人类基因组，包括约90％的复制起源，我们建议MCM6招募BLM 在复制起源处展开DNA结构（i），以促进G1/s的过渡（AIM 1）和（ii） 基因组调节不受干扰的S相过程中的复制组进展（AIM 2）。我们已经隔离了一组 专门与G1或S相中的MCM6相互作用的BLM突变体，或两者兼而有 BLM/MCM6相互作用在G1和S期间的功能，并确定与复制相关的有丝分裂 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 BLM解开G4的机制（AIM 3）。完成这些研究将描述一个主要的新功能 除了其在DNA双链破裂修复中的作用外，BLM不受干扰的DNA复制 复制叉在DNA损伤后重新启动，并确定其g4的机理。我们的发现会 在理解防止染色体不稳定的机制方面提供了重大进步 不受干扰的细胞，并提高我们对染色体破裂综合征和癌症的理解 倾向。