Replication fork stalling and checkpoint activation by a PKD1 locus mirror repeat polypurine-polypyrimidine (Pu-Py) tract.
Replication fork stalling and checkpoint activation by a PKD1 locus mirror repeat polypurine-polypyrimidine (Pu-Py) tract.
复制标题
PKD1 位点镜像重复聚嘌呤-聚嘧啶 (Pu-Py) 束导致复制叉停滞和检查点激活。
DOI：
10.1074/jbc.m112.402503
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发表时间：
2012
期刊：
影响因子：
--
通讯作者：
Leffak,Michael
中科院分区：
文献类型：
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作者：
Liu,Guoqi;Myers,Sheré;Chen,Xiaomi;Bissler,JohnJ;Sinden,RichardR;Leffak,Michael
DNA序列容易形成非规范结构(发夹、三联体、G-四联体)，导致DNA复制分叉停滞，激活DNA损伤反应，并代表与人类疾病相关的基因组不稳定的热点。人多囊肾病(PKD)内含子21的88个碱基的不对称多嘌呤-多嘧啶(Pu-Py)镜像重复序列在体外形成非B-DNA二级结构。我们表明，在体外和体内复制过程中，PKD1镜像重复也会导致方向相关的叉子停滞。当与c-mycreplicator一起整合在HeLa基因组中的异位染色体位置时，Pu-Py镜像重复道引起了极复制叉状障碍。复制蛋白A(RPA)、RAD9、以及镜像重复序列附近的共济失调毛细血管扩张和RAD3相关(ATR)检查点蛋白结合增加表明，DNA损伤反应在复制叉失速时被激活。此外，复制的近端c-mycorigin不是引起定向依赖的检查点激活所必需的。表达复制分叉障碍的细胞表现出结构性的Chk1磷酸化和持续生长，即检查点适应。切除Pu-Py镜像重复区域可以消除DNA损伤反应。在表达复制分叉屏障的细胞中对Chk1磷酸化的适应可能允许突变的积累，否则这些突变将被DNA损伤反应修复。
DNA sequences prone to forming noncanonical structures (hairpins, triplexes, G-quadruplexes) cause DNA replication fork stalling, activate DNA damage responses, and represent hotspots of genomic instability associated with human disease. The 88-bp asymmetric polypurine-polypyrimidine (Pu-Py) mirror repeat tract from the human polycystic kidney disease (PKD1) intron 21 forms non-B DNA secondary structuresin vitro. We show that thePKD1mirror repeat also causes orientation-dependent fork stalling during replicationin vitroandin vivo. When integrated alongside the c-mycreplicator at an ectopic chromosomal site in the HeLa genome, the Pu-Py mirror repeat tract elicits a polar replication fork barrier. Increased replication protein A (RPA), Rad9, and ataxia telangiectasia- and Rad3-related (ATR) checkpoint protein binding near the mirror repeat sequence suggests that the DNA damage response is activated upon replication fork stalling. Moreover, the proximal c-mycorigin of replication was not required to cause orientation-dependent checkpoint activation. Cells expressing the replication fork barrier display constitutive Chk1 phosphorylation and continued growth,i.e.checkpoint adaptation. Excision of the Pu-Py mirror repeat tract abrogates the DNA damage response. Adaptation to Chk1 phosphorylation in cells expressing the replication fork barrier may allow the accumulation of mutations that would otherwise be remediated by the DNA damage response.