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

基本信息

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
项目类别：
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

项目摘要

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会导致布卢姆综合症。极端的癌症风险、身材矮小以及 25 岁的平均预期寿命，目前，染色体断裂和其他细胞缺陷可以通过 BLM 活性的缺乏来解释。 DNA 损伤反应和同源重组在本提案中，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着关键作用该假设基于广泛的初步数据，包括无偏见的筛选。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 中有两个不同的结合位点。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。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，这使我们捕获了 BLM 的需要与 Mcm6 相连，将 BLM 的 ATP 酶/解旋酶活性限制在连同 BLM 解开 G-四链体 (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 期调节复制体进展（目标 2）。 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此外，我们将使用生物物理方法和分子动力学模拟来确定缺陷。 BLM 解开 G4 的机制（目标 3）将描绘出一个主要的新功能。 BLM 除了在 DNA 双链断裂修复和 DNA损伤后复制叉重新启动，并确定其G4解旋机制。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倾向。