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: 10457409
• 负责人: Kristina Schmidt
• 金额: $ 33.69万
• 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/10457409
• 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.

项目概要 RecQ 样 DNA 解旋酶 BLM 以其在 DNA 双螺旋响应和修复中的关键作用而闻名。 哺乳动物细胞中的链断裂。 BLM 活动中断会导致布卢姆综合症，其特征是 极端癌症风险、身材矮小和平均预期寿命为 25 岁。癌症易感性， 目前，染色体断裂和其他细胞缺陷可以通过 BLM 缺乏活性来解释。 DNA 损伤反应和同源重组。在这个提案中，我们正在测试以下假设： BLM 在 DNA 复制起始和延伸以维持染色体稳定性中发挥关键作用 在未受干扰的细胞中。该假设基于广泛的初步数据，包括对 中期 S 期蛋白质组导致发现染色质结合的 BLM 直接与 Mcm6 相互作用 染色质结合的 Mcm2-7 亚基。值得注意的是，BLM 和 Mcm6 中两个不同的结合位点存在差异 调节 G1 和 S 期的复合物形成，并破坏 S 期的 BLM/Mcm6 相互作用，但是 不在G1，导致超正常的DNA复制速度。 DNA复制速度异常加速 超过安全限度正在成为一种导致 DNA 损伤并杀死某些类型癌症的机制 细胞。我们的初步研究结果表明，BLM/Mcm6 相互作用充当一种新型负调节因子 人类细胞中的 DNA 复制。缺乏 BLM 的细胞不会表现出更高的复制速度 表明复制的加速需要 BLM 蛋白，这使我们推测 BLM 需要 与 Mcm6 相连，将 BLM 的 ATP 酶/解旋酶活性限制在 复制体。加上 BLM 解开 G 四联体 (G4) 的能力及其在整个过程中的存在 人类基因组，包括约 90% 的复制起点，我们建议 Mcm6 招募 BLM 来 (i) 在复制起点展​​开 DNA 结构，以促进 G1/S 转变（目标 1）和 (ii) 在整个复制过程中 基因组在不受干扰的 S 期期间调节复制体进展（目标 2）。我们隔离了一组 BLM 突变体特别无法与 G1 期或 S 期或两者中的 Mcm6 相互作用，以识别单独的 BLM/Mcm6 相互作用在 G1 和 S 期的功能并确定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 的 G4 展开机制（目标 3）。完成这些研究将为 BLM 除了在 DNA 双链断裂修复和修复中的既定作用外，还能在不受干扰的 DNA 复制中发挥作用 DNA损伤后复制叉重新启动，并确定其G4解旋机制。我们的研究结果将 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了解 倾向。