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

项目总结/摘要 由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 在体细胞组织中，以及在配子发生干细胞中发生的新生和遗传性遗传疾病， 减数分裂复制失败的主要致突变结果是结构变异体（SV）的形成，尤其是拷贝数的减少。 数量变异（CNVs），在单个突变步骤中产生基因组内容的巨大变化。基本 在导致SV形成的DNA修复机制方面存在知识空白。虽然多个 可能涉及的机制，从模板转换，断裂诱导复制（BIR）， 其他形式的双链断裂（DSB）修复已经被提出来占人类的大部分。 CNVs，但缺乏直接的实验证据。我们先前的工作表明，不完全复制会导致 人类细胞中CNV的频率较高，大的转录基因中有热点，对应于常见的 脆性位点（CFSs），其提供用于表征SV形成机制的模型系统。 最近的文献已经揭示了很多关于促进正确完成的损伤反应途径 复制。其中一个发现是有丝分裂DNA合成（MiDAS），其中S中失败的复制被一个 复制的保守形式直到有丝分裂时才被激活。作为BIR样途径，MiDAS准确性被认为是 因此，MiDAS和CFS表达之间的时间关联表明， 与SV形成的机械联系。我们的主要目标是探索复制拯救， 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热点基因座处的观察结果对全基因组SV形成的可扩展性。我们的中央 假设是复制拯救期间发生热点CNV形成，无论是通过MiDAS还是替代方法 在MiDAS通路中，特别是θ介导的末端连接（TMEJ）。一个驱动原理是，我们必须监控SV 形成在真实的时间作为一个主要的实验成果，我们一直致力于做的事情。 因此，我们的方法将应用我们最近的技术进步，直接检测罕见的SV连接， 实验样本，为长期存在的关于人类SV起源的问题提供答案。 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， 复制应激后形成结构变异体;（2）建立复制拯救和DNA修复途径 产生结构变异连接;和（3）将有丝分裂SV形成机制从CFS扩展到整个 基因组和BRCA 2缺陷。该组合是重要的，因为它将提供直接的实验测试， 在风险基因组位点中执行SV形成的机制，并将这些发现扩展到多个基因组位点， 与体细胞和可遗传的种系诱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.