Johansson Fredrik, Lagerqvist Anne, Erixon Klaus, Jenssen Dag
Department of Genetics, Microbiology and Toxicology, Arrhenius Laboratories for Natural Sciences, Stockholm University, S-106 91 Stockholm, Sweden.
Nucleic Acids Res. 2004 Nov 10;32(20):e157. doi: 10.1093/nar/gnh154.
The capacity to rescue stalled replication forks (RFs) is important for the maintenance of cell viability and genome integrity. Here, we have developed a novel method for monitoring RF progression and the influence of DNA lesions on this process. The method is based on the principle that each RF is expected to be associated with a pair of single-stranded ends, which can be analyzed by employing strand separation in alkali. This method was applied to examine the rate of RF progression in Chinese hamster cell lines deficient in ERCC1, which is involved in nucleotide excision repair (NER), or in XRCC3, which participates in homologous recombination repair, following irradiation with ultraviolet (UV) light or exposure to benzo(a)pyrene-7,8-diol-9,10-epoxide (BPDE). The endpoints observed were cell survival, NER activity, formation of double-strand breaks and the rate of RF progression. Subsequently, we attempted to explain our observation that cells deficient in XRCC3 (irs1SF) exhibit enhanced sensitivity to UV radiation and BPDE. irs1SF cells demonstrated a capacity for NER that was comparable with wild-type AA8 cells, but the rate of RF progression was even higher than that for the wild-type AA8 cells. As expected, cells deficient in ERCC1 (UV4) showed no NER activity and were hypersensitive to both UV radiation and BPDE. The observation that cells deficient in NER displayed a pronounced delay in RF progression indicates that NER plays an important role in maintaining fork progression along damaged DNA. The elevated rate of RF progression in XRCC3-deficient cells indicates that this protein is involved in a time-consuming process which resolves stalled RFs.
挽救停滞复制叉（RFs）的能力对于维持细胞活力和基因组完整性至关重要。在此，我们开发了一种监测RF进展以及DNA损伤对该过程影响的新方法。该方法基于这样的原理：每个RF预计与一对单链末端相关联，可通过在碱性条件下进行链分离来分析。此方法用于检测在紫外线（UV）照射或接触苯并(a)芘-7,8-二醇-9,10-环氧化物（BPDE）后，缺乏参与核苷酸切除修复（NER）的ERCC1或参与同源重组修复的XRCC3的中国仓鼠细胞系中RF进展的速率。观察的终点包括细胞存活、NER活性、双链断裂的形成以及RF进展的速率。随后，我们试图解释我们的观察结果，即缺乏XRCC3的细胞（irs1SF）对UV辐射和BPDE表现出增强的敏感性。irsSF细胞表现出与野生型AA8细胞相当的NER能力，但RF进展速率甚至高于野生型AA8细胞。正如预期的那样，缺乏ERCC1的细胞（UV4）没有NER活性，并且对UV辐射和BPDE都高度敏感。缺乏NER的细胞在RF进展中表现出明显延迟的观察结果表明，NER在维持受损DNA上的叉进展中起重要作用。缺乏XRCC3的细胞中RF进展速率升高表明该蛋白参与了一个解决停滞RFs的耗时过程。
