Adaptive mutations can cause drug resistance in cancers and pathogens, and increase the tolerance of agricultural pests and diseases to chemical treatment. When and how adaptive mutations form is often hard to discern, but we have shown that adaptive copy number amplification of the copper resistance gene CUP1 occurs in response to environmental copper due to CUP1 transcriptional activation. Here we dissect the mechanism by which CUP1 transcription in budding yeast stimulates copy number variation (CNV). We show that transcriptionally stimulated CNV requires TREX-2 and Mediator, such that cells lacking TREX-2 or Mediator respond normally to copper but cannot acquire increased resistance. Mediator and TREX-2 cause replication stress by tethering transcribed loci to nuclear pores, a process known as gene gating, and transcription at the CUP1 locus causes a TREX-2-dependent accumulation of replication forks indicative of replication fork stalling. TREX-2-dependent CUP1 gene amplification occurs by a Rad52 and Rad51-mediated homologous recombination mechanism that is enhanced by histone H3K56 acetylation and repressed by Pol32, factors known to alter the frequency of template switching during break induced replication (BIR). CUP1 amplification is also critically dependent on late firing replication origins present in the CUP1 repeats, and mutations that remove or inactivate these origins strongly suppress the acquisition of copper resistance. We propose that replicative stress imposed by nuclear pore association causes replication bubbles from these origins to collapse soon after firing, leaving an epigenetic scar of H3K56 acetylation that promotes template switching during later break induced replication events. The capacity for inefficient replication origins to promote copy number variation renders certain genomic regions more fragile than others, and therefore more likely to undergo adaptive evolution through de novo gene amplification.

中文翻译：

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复制叉约束下通过原位激发来刺激自适应基因扩增

适应性突变可引起癌症和病原体的耐药性，并增加农业病虫害对化学治疗的耐受性。通常很难确定何时以及如何形成适应性突变，但是我们已经表明，由于CUP1转录激活，铜抗性基因CUP1的适应性拷贝数扩增随环境铜而发生。在这里，我们剖析了发芽酵母中CUP1转录刺激拷贝数变异（CNV）的机制。我们表明，转录刺激的CNV需要TREX-2和介体，从而使缺少TREX-2或介体的细胞对铜反应正常，但不能获得增加的抗性。介体和TREX-2通过将转录的基因座拴在核孔上而引起复制压力，这一过程称为基因门控，CUP1位点的转录会导致TREX-2依赖的复制叉积累，表明复制叉停滞。依赖TREX-2的CUP1基因扩增是通过Rad52和Rad51介导的同源重组机制发生的，该机制通过组蛋白H3K56乙酰化作用增强，并被Pol32抑制，Pol32是已知的在断裂诱导复制（BIR）过程中改变模板切换频率的因素。CUP1扩增还严重依赖于CUP1重复序列中存在的后期复制起点，而去除或使这些起点失活的突变强烈抑制了铜抗性的获得。我们认为，核孔缔合所施加的复制应力会导致这些起源的复制气泡在发射后很快崩溃，留下H3K56乙酰化的表观遗传疤痕，在以后的断裂诱导的复制事件中促进模板转换。无效复制起点促进拷贝数变异的能力使得某些基因组区域比其他基因组区域更脆弱，因此更有可能通过从头基因扩增进行适应性进化。