Yu Chuanhe, Gan Haiyun, Han Junhong, Zhou Zhi-Xiong, Jia Shaodong, Chabes Andrei, Farrugia Gianrico, Ordog Tamas, Zhang Zhiguo
Department of Biochemistry and Molecular Biology, Mayo Clinic College of Medicine, Rochester, MN 55905, USA.
Department of Medical Biochemistry and Biophysics, Umeå University, 901 87 Umeå, Sweden.
Mol Cell. 2014 Nov 20;56(4):551-63. doi: 10.1016/j.molcel.2014.09.017. Epub 2014 Oct 23.
In eukaryotic cells, DNA replication proceeds with continuous synthesis of leading-strand DNA and discontinuous synthesis of lagging-strand DNA. Here we describe a method, eSPAN (enrichment and sequencing of protein-associated nascent DNA), which reveals the genome-wide association of proteins with leading and lagging strands of DNA replication forks. Using this approach in budding yeast, we confirm the strand specificities of DNA polymerases delta and epsilon and show that the PCNA clamp is enriched at lagging strands compared with leading-strand replication. Surprisingly, at stalled forks, PCNA is unloaded specifically from lagging strands. PCNA unloading depends on the Elg1-containing alternative RFC complex, ubiquitination of PCNA, and the checkpoint kinases Mec1 and Rad53. Cells deficient in PCNA unloading exhibit increased chromosome breaks. Our studies provide a tool for studying replication-related processes and reveal a mechanism whereby checkpoint kinases regulate strand-specific unloading of PCNA from stalled replication forks to maintain genome stability.
在真核细胞中，DNA复制以连续合成前导链DNA和不连续合成后随链DNA的方式进行。在此，我们描述了一种方法，即eSPAN（蛋白质相关新生DNA的富集与测序），它揭示了全基因组范围内蛋白质与DNA复制叉前导链和后随链的关联。在芽殖酵母中使用这种方法，我们证实了DNA聚合酶δ和ε的链特异性，并表明与前导链复制相比，增殖细胞核抗原（PCNA）夹子在后随链上富集。令人惊讶的是，在停滞的复制叉处，PCNA特异性地从后随链上卸载。PCNA卸载依赖于含Elg1的替代复制因子C（RFC）复合物、PCNA的泛素化以及检查点激酶Mec1和Rad53。缺乏PCNA卸载功能的细胞表现出染色体断裂增加。我们的研究为研究复制相关过程提供了一种工具，并揭示了一种机制，即检查点激酶调节PCNA从停滞的复制叉上进行链特异性卸载以维持基因组稳定性。
