title: Replication-fork stalling and processing at a single psoralen interstrand crosslink in Xenopus egg extracts.
abstract: Interstrand crosslink (ICL)-inducing agents block the separation of the two DNA strands. They prevent transcription and replication and are used in clinics for the treatment of cancer and skin diseases. Here, we have introduced a single psoralen ICL at a specific site in plasmid DNA using a triplex-forming-oligonucleotide (TFO)-psoralen conjugate and studied its repair in Xenopus egg extracts that support nuclear assembly and replication of plasmid DNA. Replication forks arriving from either side stalled at the psoralen ICL. In contrast to previous observations with other ICL-inducing agents, the leading strands advanced up to the lesion without any prior pausing. Subsequently, incisions were introduced on one parental strand on both sides of the ICL. These incisions could be detected whether one or both forks reached the ICL. Using small molecule inhibitors, we found that the ATR-Chk1 pathway, but not the ATM-Chk2 pathway, stimulated both the incision step and the subsequent processing of the broken replication intermediates. Our results highlight both similarities and differences in fork stalling and repair induced by psoralen and by other ICL-forming agents.
authors.affiliation: Institut de Biologie de l'Ecole Normale Supérieure, CNRS UMR 8197-Inserm U1024, Paris, France.
authors.forename: Cyrille
authors.initials: C
authors.lastname: Le Breton
authors.forename: Magali
authors.initials: M
authors.lastname: Hennion
authors.forename: Paola B
authors.initials: PB
authors.lastname: Arimondo
authors.forename: Olivier
authors.initials: O
authors.lastname: Hyrien
mesh_terms: D000818:Animals; D001483:Base Sequence; D002457:Cell Extracts; D000071877:Checkpoint Kinase 1; D003432:Cross-Linking Reagents / Q000494:pharmacology*; D004247:DNA / Q000378:metabolism*; D004261:DNA Replication / Q000187:drug effects*; D015180:Electrophoresis, Gel, Two-Dimensional; D005363:Ficusin / Q000494:pharmacology*; D008954:Models, Biological; D008969:Molecular Sequence Data; D009841:Oligonucleotides / Q000235:genetics; D010063:Ovum / Q000166:cytology / Q000187:drug effects* / Q000378:metabolism*; D010957:Plasmids / Q000235:genetics / Q000302:isolation & purification; D047428:Protein Kinase Inhibitors / Q000494:pharmacology; D011494:Protein Kinases / Q000378:metabolism; D017346:Protein Serine-Threonine Kinases / Q000037:antagonists & inhibitors / Q000378:metabolism; D014981:Xenopus / Q000378:metabolism*; D029867:Xenopus Proteins
chemical_list: D002457:Cell Extracts; D003432:Cross-Linking Reagents; D009841:Oligonucleotides; D047428:Protein Kinase Inhibitors; D029867:Xenopus Proteins; D004247:DNA; D011494:Protein Kinases; D000071877:Checkpoint Kinase 1; C000605875:Chek1 protein, Xenopus; D017346:Protein Serine-Threonine Kinases; D005363:Ficusin