Checkpoint signaling from a single DNA interstrand crosslink.
DNA interstrand crosslinks (ICLs) are the most toxic lesions induced by chemotherapeutic agents such as mitomycin C and cisplatin. By covalently linking both DNA strands, ICLs prevent DNA melting, transcription, and replication. Studies on ICL signaling and repair have been limited, because these drugs generate additional DNA lesions that trigger checkpoint signaling. Here, we monitor sensing, signaling from, and repairing of a single site-specific ICL in cell-free extract derived from Xenopus eggs and in mammalian cells. Notably, we demonstrate that ICLs trigger a checkpoint response independently of origin-initiated DNA replication and uncoupling of DNA polymerase and DNA helicase. The Fanconi anemia pathway acts upstream of RPA-ATR-Chk1 to generate the ICL signal. The system also repairs ICLs in a reaction that involves extensive, error-free DNA synthesis. Repair occurs by both origin-dependent and origin-independent mechanisms. Our data suggest that cell sensitivity to crosslinking agents results from both checkpoint and DNA repair defects.
Ben-Yehoyada
Merav
M
Institute for Cancer Genetics, Department of Genetics and Development, Columbia University, New York, NY 10032, USA.
Wang
Lily C
LC
Kozekov
Ivan D
ID
Rizzo
Carmelo J
CJ
Gottesman
Max E
ME
Gautier
Jean
J
D000477:Alkylating Agents / Q000494:pharmacology; D000818:Animals; D064007:Ataxia Telangiectasia Mutated Proteins; D002453:Cell Cycle / Q000235:genetics*; D018797:Cell Cycle Proteins / Q000378:metabolism; D049109:Cell Proliferation; D000071877:Checkpoint Kinase 1; D004247:DNA / Q000096:biosynthesis / Q000737:chemistry / Q000378:metabolism*; D004249:DNA Damage; D004265:DNA Helicases / Q000378:metabolism; D004260:DNA Repair; D004261:DNA Replication; D004259:DNA-Directed DNA Polymerase / Q000378:metabolism; D052217:Fanconi Anemia Complementation Group A Protein / Q000378:metabolism; D052236:Fanconi Anemia Complementation Group D2 Protein / Q000378:metabolism; D006367:HeLa Cells; D006801:Humans; D009690:Nucleic Acid Conformation; D011494:Protein Kinases / Q000378:metabolism; D017346:Protein Serine-Threonine Kinases / Q000378:metabolism; D011994:Recombinant Proteins / Q000378:metabolism; D018741:Replication Origin; D051716:Replication Protein A / Q000378:metabolism; D015398:Signal Transduction / Q000235:genetics*; D013997:Time Factors; D014162:Transfection; D029867:Xenopus Proteins; D014982:Xenopus laevis
D000477:Alkylating Agents; D018797:Cell Cycle Proteins; C498471:FANCA protein, human; C499905:FANCD2 protein, human; D052217:Fanconi Anemia Complementation Group A Protein; D052236:Fanconi Anemia Complementation Group D2 Protein; D011994:Recombinant Proteins; D051716:Replication Protein A; D029867:Xenopus Proteins; D004247:DNA; D011494:Protein Kinases; C576597:ATR protein, human; D064007:Ataxia Telangiectasia Mutated Proteins; C000605867:CHEK1 protein, human; D000071877:Checkpoint Kinase 1; C000605875:Chek1 protein, Xenopus; D017346:Protein Serine-Threonine Kinases; D004259:DNA-Directed DNA Polymerase; D004265:DNA Helicases