title: DNA interstrand crosslinks induce a potent replication block followed by formation and repair of double strand breaks in intact mammalian cells.
abstract: DNA interstrand crosslinks (ICLs) are highly toxic lesions that covalently link both strands of DNA and distort the DNA helix. Crosslinking agents have been shown to stall DNA replication and failure to repair ICL lesions before encountered by replication forks may induce severe DNA damage. Most knowledge of the ICL repair process has been revealed from studies in bacteria and cell extracts. However, for mammalian cells the process of ICL repair is still unclear and conflicting data exist. In this study we have explored the fate of psoralen-induced ICLs during replication, by employing intact mammalian cells and novel techniques. By comparative studies distinguishing between effects by monoadducts versus ICLs, we have been able to link the block of replication to the ICLs induction. We found that the replication fork was equally blocked by ICLs in wild-type cells as in cells deficient in ERCC1/XPF and XRCC3. The formation of ICL induced double strand breaks (DSBs), detected by formation of 53PB1 foci, was equally induced in the three cell lines suggesting that these proteins are involved at a later step of the repair process. Furthermore, we found that forks blocked by ICLs were neither bypassed, restarted nor restored for several hours. We propose that this process is different from that taking place following monoadduct induction by UV-light treatment where replication bypass is taking place as an early step. Altogether our findings suggest that restoration of an ICL blocked replication fork, likely initiated by a DSB occurs relatively rapidly at a stalled fork, is followed by restoration, which seems to be a rather slow process in intact mammalian cells.
authors.affiliation: Department of Genetics, Microbiology and Toxicology, Arrhenius Laboratories of Natural Science, Stockholm University, S-106 91 Stockholm, Sweden.
authors.forename: Daniel
authors.initials: D
authors.lastname: Vare
authors.forename: Petra
authors.initials: P
authors.lastname: Groth
authors.forename: Rickard
authors.initials: R
authors.lastname: Carlsson
authors.forename: Fredrik
authors.initials: F
authors.lastname: Johansson
authors.forename: Klaus
authors.initials: K
authors.lastname: Erixon
authors.forename: Dag
authors.initials: D
authors.lastname: Jenssen
mesh_terms: D000818:Animals; D016466:CHO Cells; D002470:Cell Survival; D006224:Cricetinae; D003432:Cross-Linking Reagents / Q000009:adverse effects*; D053903:DNA Breaks, Double-Stranded; D004260:DNA Repair; D004261:DNA Replication; D004268:DNA-Binding Proteins / Q000235:genetics; D005363:Ficusin / Q000009:adverse effects*; D011564:Furocoumarins / Q000009:adverse effects; D020128:Inhibitory Concentration 50; D008322:Mammals; D011995:Recombination, Genetic; D018741:Replication Origin; D014466:Ultraviolet Rays
chemical_list: D003432:Cross-Linking Reagents; D004268:DNA-Binding Proteins; D011564:Furocoumarins; C110901:X-ray repair cross complementing protein 3; C011659:angelicin; D005363:Ficusin