Cell cycle timing and molecular mechanisms of structural variant formation following incomplete replication

不完全复制后结构变异形成的细胞周期时间和分子机制

基本信息

批准号：
10656861
负责人：
THOMAS W GLOVER
金额：
$ 51.65万
依托单位：
UNIVERSITY OF MICHIGAN AT ANN ARBOR
依托单位国家：
美国
项目类别：
财政年份：
2023
资助国家：
美国
起止时间：
2023-04-12 至 2027-03-31
项目状态：
未结题

来源：
https://reporter.nih.gov/project-details/10656861
关键词：
Automobile Driving BRCA2 gene Biological Models CRISPR/Cas technology Cell Culture Techniques Cell Cycle Cell Cycle Stage Cell Fraction Cell Lineage Cells Chemicals Chromosomal Rearrangement Chromosome Fragile Sites Copy Number Polymorphism DNA DNA Repair DNA Repair Pathway DNA Synthesis Inhibition DNA biosynthesis Data Dedications Double Strand Break Repair Event FANCD2 protein Failure Frequencies Functional disorder Genes Genetic Diseases Genetic Transcription Genome Genomic Segment Genomic approach Genomics Germ-Line Mutation Goals Hereditary Disease Heritability Human Human Genetics Human Genome Knock-out Knowledge Link Literature Malignant Neoplasms Mediating Meiosis Methods Mission Mitosis Mitotic Mitotic M Phase Modeling Molecular Monitor Mutagenesis Mutation Outcome Pathway interactions Pattern Public Health RAD52 gene Reporting Resolution Risk Role S phase Sampling Small Interfering RNA Sorting Stress System Technology Testing Time Tissues United States National Institutes of Health Variant Work genetic information genome-wide genomic locus insight mutant novel preservation prevent replication stress response small hairpin RNA stem cells transmission process whole genome

项目摘要

Project Summary / Abstract Mutagenesis resulting from replication failure is a direct cause of tissue dysfunction and cancer when it occurs in somatic tissues and of de novo and inherited genetic diseases when it occurs in gametogenic stem cells or meiosis. A primary mutagenic outcome of replication failure is structural variant (SV) formation, especially copy number variants (CNVs), which create large changes in genomic content in single mutational steps. Fundamental gaps in knowledge exist regarding the DNA repair mechanisms that lead to SV formation. While multiple mechanisms may be involved, models that derive from template switching, break-induced replication (BIR), and other forms of double-strand break (DSB) repair have been forwarded to account for a large proportion of human CNVs but lack direct experimental evidence. Our prior work has shown that incomplete replication leads to a high frequency of CNVs in human cells, with hotspots in large, transcribed genes corresponding to common fragile sites (CFSs) that provide a model system for characterizing SV formation mechanisms. Recent literature has revealed much about the damage response pathways that promote proper completion of replication. One finding was Mitotic DNA Synthesis (MiDAS), where failed replication in S is rescued by a conservative form of replication activated as late as mitosis. As a BIR-like pathway, MiDAS accuracy is thought to be low such that the temporal association between MiDAS and CFS expression suggests a potential mechanistic link to SV formation. Our major goals are to explore the relationships between replication rescue, end-joining, BIR, other forms of DSB repair, and SV formation, how CFS expression relates to CNV formation, and how extensible observations at CFS/CNV hotspot loci are to SV formation genome wide. Our central hypothesis is that hotspot CNV formation occurs during replication rescue, either via MiDAS or an alternative pathway to MiDAS, notably, theta-mediated end joining (TMEJ). A driving rationale is that we must monitor SV formation in real time as a primary experimental outcome, something we have been uniquely dedicated to doing. Our approach will therefore apply our recent technology advances for directly detecting rare SV junctions in experimental samples to provide answers to longstanding questions about the origins of human SVs. We will address our goals through three specific aims to (1) Identify the precise cell cycle stage(s) when structural variants form following replication stress; (2) Establish the replication rescue and DNA repair pathways that create structural variant junctions; and (3) Extend mitotic SV formation mechanisms from CFSs to the whole genome and BRCA2 deficiency. The combination is significant as it will provide direct experimental tests of the mechanisms that execute SV formation in at-risk genomic loci and extend those findings to multiple genomic regions and cell lineages relevant to both somatic and heritable germline mutagenesis.

项目概要/摘要复制失败引起的突变是组织功能障碍和癌症发生的直接原因当它发生在配子干细胞或减数分裂。复制失败的主要诱变结果是结构变异（SV）的形成，尤其是复制数字变异（CNV），在单个突变步骤中使基因组内容产生巨大变化。基本的关于导致 SV 形成的 DNA 修复机制存在知识空白。虽然多个可能涉及机制、源自模板切换、断裂诱导复制 (BIR) 的模型以及其他形式的双链断裂（DSB）修复已在人类中占很大比例 CNV但缺乏直接的实验证据。我们之前的工作表明，不完整的复制会导致人类细胞中出现高频率的 CNV，热点位于与常见基因相对应的大型转录基因中脆弱位点 (CFS) 提供了表征 SV 形成机制的模型系统。最近的文献揭示了许多有关促进正确完成的损伤反应途径的信息的复制。一项发现是有丝分裂 DNA 合成 (MiDAS)，其中 S 中的复制失败由一个保守的复制形式晚至有丝分裂才被激活。作为类似 BIR 的通路，MiDAS 的准确性被认为低，使得 MiDAS 和 CFS 表达之间的时间关联表明潜在的与 SV 形成的机制联系。我们的主要目标是探索复制拯救之间的关系，末端连接、BIR、其他形式的 DSB 修复和 SV 形成、CFS 表达如何与 CNV 形成相关，以及 CFS/CNV 热点基因座的观察结果如何扩展到基因组范围内的 SV 形成。我们的中央假设热点 CNV 形成发生在复制救援期间，通过 MiDAS 或替代方案 MiDAS 的途径，特别是 theta 介导的末端连接 (TMEJ)。一个重要的理由是我们必须监控 SV 实时形成作为主要实验结果，这是我们一直致力于做的事情。因此，我们的方法将应用我们最新的技术进步来直接检测罕见的 SV 连接实验样本为有关人类 SV 起源的长期问题提供答案。我们将通过三个具体目标来实现我们的目标：(1) 确定精确的细胞周期阶段复制压力后形成结构变异； (2) 建立复制拯救和DNA修复途径创建结构变异连接； (3) 将有丝分裂 SV 形成机制从 CFS 扩展到整个基因组和 BRCA2 缺陷。这一组合意义重大，因为它将提供直接的实验测试在有风险的基因组位点中执行 SV 形成并将这些发现扩展到多个基因组的机制与体细胞和可遗传种系突变相关的区域和细胞谱系。