A method to monitor replication fork progression in mammalian cells: nucleotide excision repair enhances and homologous recombination delays elongation along damaged DNA.
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.
Johansson
Fredrik
F
Department of Genetics, Microbiology and Toxicology, Arrhenius Laboratories for Natural Sciences, Stockholm University, S-106 91 Stockholm, Sweden.
Lagerqvist
Anne
A
Erixon
Klaus
K
Jenssen
Dag
D
D015123:7,8-Dihydro-7,8-dihydroxybenzo(a)pyrene 9,10-oxide / Q000633:toxicity; D000818:Animals; D016466:CHO Cells; D002460:Cell Line; D006224:Cricetinae; D003412:Cricetulus; D018736:DNA Adducts / Q000378:metabolism; D004249:DNA Damage; D004260:DNA Repair; D004261:DNA Replication; D004268:DNA-Binding Proteins / Q000235:genetics; D004720:Endonucleases / Q000235:genetics; D005821:Genetic Techniques; D007700:Kinetics; D009154:Mutation; D011995:Recombination, Genetic; D014466:Ultraviolet Rays
D018736:DNA Adducts; D004268:DNA-Binding Proteins; C110901:X-ray repair cross complementing protein 3; D015123:7,8-Dihydro-7,8-dihydroxybenzo(a)pyrene 9,10-oxide; D004720:Endonucleases