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

项目摘要/摘要 复制失败导致的突变是导致组织功能障碍和癌症的直接原因 在体细胞组织和新生和遗传性遗传病中发生在配子发育干细胞或 减数分裂。复制失败的主要诱变结果是结构变异(SV)的形成，尤其是拷贝 数量变异(CNV)，在单个突变步骤中造成基因组内容的巨大变化。基本原理 关于导致SV形成的DNA修复机制存在知识空白。虽然有多个 可能涉及机制、从模板切换派生的模型、中断诱导复制(BIR)以及 其他形式的双链断裂(DSB)修复已经被提出，占人类的很大比例 但缺乏直接的实验证据。我们之前的工作已经表明，不完整的复制会导致 在人类细胞中CNV的频率很高，在大的、转录的基因中有热点，对应于常见的 脆性部位(CFSS)，提供了描述SV形成机制的模型系统。 最近的文献揭示了许多关于促进正常完成的损伤反应途径 复制的结果。其中一个发现是有丝分裂DNA合成(MIDAS)，在这种情况下，S的失败复制被一种 保守形式的复制在有丝分裂后期才被激活。作为一种类似BIR的途径，MIDAS被认为是准确的 低到MIDAS和CFS表达之间的时间关联暗示了潜在的 与SV队形的机械连接。我们的主要目标是探索复制救援和 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， 以及在CFS/CNV热点位点的观察对SV形成基因组范围的可扩充性。我们的中央 假设在复制抢救过程中，通过Midas或替代方法形成热点CNV 通往MIDAS的途径，特别是theta介导的末端连接(TMEJ)。一个驱动的理由是，我们必须监控SV 实时形成作为主要的实验结果，这是我们唯一致力于做的事情。 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，以提供关于人类SVS起源的长期问题的答案。 我们将通过三个具体目标来解决我们的目标：(1)确定准确的细胞周期阶段(S)，当 复制胁迫后形成结构变异；(2)建立复制修复和DNA修复途径 这创造了结构不同的连接；以及(3)将有丝分裂SV的形成机制从CFSS扩展到整个 基因组与BRCA2缺乏症这一组合意义重大，因为它将提供对 在高危基因组座位上执行SV形成并将这些发现扩展到多个基因组的机制 与体细胞和可遗传生殖系突变相关的区域和细胞谱系。

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.