The mode of action of 1-beta-D-arabinofuranosylcytosine in inhibiting DNA repair; new evidence using a sensitive assay for repair DNA synthesis and ligation in permeable cells.
Mammalian cells permeabilised by treatment with saponin are capable of UV excision repair. We have developed an assay system which permits measurement of the later stages of repair, i.e. repair synthesis and ligation. Incomplete repair sites are accumulated in UV-irradiated cells by incubating them with DNA synthesis inhibitors hydroxyurea and aphidicolin. On removal of the inhibitors at the time of permeabilisation, these incomplete sites, detected as DNA breaks, are rapidly ligated in a reaction that requires deoxyribonucleoside triphosphates and is blocked by aphidicolin. Thus ligation is possible only after a significant amount of DNA synthesis. We have used the assay to clarify the mode of inhibition of DNA repair by 1-beta-D-arabinofuranosylcytosine (ara C), another DNA polymerase inhibitor. It is well known that incomplete repair sites accumulated in whole cells with ara C are ligated at a slow rate, if at all. The hypothesis that ara C blocks or reduces further polymerisation after its incorporation into repair patches is disproved by our demonstration that, in permeable cells, the accumulated DNA breaks are ligated very rapidly. The likely explanation of the action of ara C is that, once phosphorylated, it remains in the cell as ara CTP and continues to inhibit polymerisation through competition with dCTP; in permeable cells, it readily leaks out.
Gedik
C M
CM
Department of Molecular and Cell Biology, Marischal College, University of Aberdeen, Great Britain.
Collins
A R
AR
D016590:Aphidicolin; D001667:Binding, Competitive; D002463:Cell Membrane Permeability / Q000187:drug effects; D003561:Cytarabine / Q000494:pharmacology*; D004247:DNA / Q000096:biosynthesis* / Q000528:radiation effects; D004249:DNA Damage; D004260:DNA Repair / Q000187:drug effects*; D004224:Diterpenes / Q000494:pharmacology; D006367:HeLa Cells / Q000528:radiation effects; D006801:Humans; D006918:Hydroxyurea / Q000494:pharmacology; D019384:Nucleic Acid Synthesis Inhibitors; D014466:Ultraviolet Rays
D004224:Diterpenes; D019384:Nucleic Acid Synthesis Inhibitors; D003561:Cytarabine; D016590:Aphidicolin; D004247:DNA; D006918:Hydroxyurea