Replication stress at microsatellites causes DNA double-strand breaks and break-induced replication
Replication stress at microsatellites causes DNA double-strand breaks and break-induced replication
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DOI：
10.1074/jbc.ra120.013495
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发表时间：
2020-11-06
影响因子：
4.8
通讯作者：
Leffak, Michael
中科院分区：
文献类型：
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作者：
Gadgil, Rujuta Yashodhan;Romer, Eric J.;Leffak, Michael
短串联重复的DNA序列，称为微卫星，在人类基因组中很丰富。这些微卫星表现出长度不稳定性和DNA双链断裂（DSB）的敏感性，由于它们倾向于形成稳定的非B DNA结构。复制依赖性微卫星DSB与人类发育疾病和癌症中的基因组不稳定性特征有关。为了探索微卫星DSB的原因和后果，我们设计了一个双荧光报告系统来检测DSB在扩展（CTG/CAG）（n）和多嘌呤/多嘧啶（Pu/Py）镜像重复结构旁边的c-myc复制起点整合在一个单一的异位染色体位点。（CTG/CAG）（100）微卫星附近的限制性切割导致侧翼AluY元件之间的同源定向单链退火和报告基因缺失，可以通过流式细胞术检测到。然而，在不存在限制性切割的情况下，内源性和外源性复制应激物在（CTG/CAG）（100）和Pu/Py微卫星处诱导DSB。DSB映射到（CTG）（100）滞后链模板下游边缘的狭窄区域。(CTG/CAG）（n）染色体脆性依赖于重复序列长度，而（Pu/Py）微卫星的不稳定性依赖于复制极性。引人注目的是，限制性产生的DSB和复制依赖性DSB的修复机制不同。DNA损伤反应蛋白的敲低增加（Rad 18、聚合酶（Pol）eta、Pol kappa）或降低（Mus 81）（CTG/CAG）（100）微卫星对复制应激的敏感性。复制应激和异位（CTG/CAG）（100）微卫星的DSB导致断裂诱导的复制和侧翼胸苷激酶基因的高频突变。我们的研究结果表明，非B结构倾向的微卫星容易受到复制依赖的DSB，导致基因组不稳定。
Short tandemly repeated DNA sequences, termed microsatellites, are abundant in the human genome. These microsatellites exhibit length instability and susceptibility to DNA double-strand breaks (DSBs) due to their tendency to form stable non-B DNA structures. Replication-dependent microsatellite DSBs are linked to genome instability signatures in human developmental diseases and cancers. To probe the causes and consequences of microsatellite DSBs, we designed a dual-fluorescence reporter system to detect DSBs at expanded (CTG/CAG)(n) and polypurine/polypyrimidine (Pu/Py) mirror repeat structures alongside the c-myc replication origin integrated at a single ectopic chromosomal site. Restriction cleavage near the (CTG/CAG)(100) microsatellite leads to homology-directed single-strand annealing between flanking AluY elements and reporter gene deletion that can be detected by flow cytometry. However, in the absence of restriction cleavage, endogenous and exogenous replication stressors induce DSBs at the (CTG/CAG)(100) and Pu/Py microsatellites. DSBs map to a narrow region at the downstream edge of the (CTG)(100) lagging-strand template. (CTG/CAG)(n) chromosome fragility is repeat length-dependent, whereas instability at the (Pu/Py) microsatellites depends on replication polarity. Strikingly, restriction-generated DSBs and replication-dependent DSBs are not repaired by the same mechanism. Knockdown of DNA damage response proteins increases (Rad18, polymerase (Pol) eta, Pol kappa) or decreases (Mus81) the sensitivity of the (CTG/CAG)(100) microsatellites to replication stress. Replication stress and DSBs at the ectopic (CTG/CAG)(100) microsatellite lead to break-induced replication and high-frequency mutagenesis at a flanking thymidine kinase gene. Our results show that non-B structure-prone microsatellites are susceptible to replication-dependent DSBs that cause genome instability.