Structure of repaired sites in human DNA synthesized in the presence of inhibitors of DNA polymerases alpha and beta in human fibroblasts.
Excision repair of ultraviolet damage in human fibroblasts was partially inhibited by drugs that block DNA polymerases alpha or beta (cytosine arabinoside, aphidicolin and dideoxythymidine) causing a reduction in unscheduled synthesis and an accumulation of single-strand breaks. The strand breaks accumulated in the presence of aphidicolin could be resealed within 30 min after removal of the drug, but those accumulated by cytosine arabinoside took many hours. Digestion of repaired DNA with exonuclease III or S1 nuclease revealed that even the highest concentration of polymerase inhibitors, singly or in combination, that produced maximal accumulation of single-strand breaks only blocked 37-86% of repair sites. Use of single-strand break frequencies to measure the number of repair events can therefore be in error by as much as a factor of 3. The blocked patches with free 3'OH termini were, on average, 22% of normal length, corresponding to between 6 and 17 bases (assuming a normal patch of 25-75 bases in length). Patches that remained unsealed in vivo were also resistant to sealing by T4 ligase in vitro. The data are more consistent with a mechanism of repair in which long single-strand gaps are first made by excision enzymes and subsequently filled in by DNA polymerase alpha. Strand displacement or nick translation mechanisms seem unlikely.
D016590:Aphidicolin; D002478:Cells, Cultured; D003561:Cytarabine / Q000494:pharmacology*; D004256:DNA Polymerase I / Q000037:antagonists & inhibitors*; D004257:DNA Polymerase II / Q000037:antagonists & inhibitors*; D004260:DNA Repair; D004261:DNA Replication / Q000187:drug effects*; D015224:Dideoxynucleosides; D004224:Diterpenes / Q000494:pharmacology*; D005347:Fibroblasts / Q000187:drug effects / Q000378:metabolism; D006801:Humans; D007700:Kinetics; D019384:Nucleic Acid Synthesis Inhibitors; D012867:Skin / Q000187:drug effects / Q000378:metabolism; D013936:Thymidine / Q000031:analogs & derivatives* / Q000494:pharmacology
D015224:Dideoxynucleosides; D004224:Diterpenes; D019384:Nucleic Acid Synthesis Inhibitors; D003561:Cytarabine; C019336:2',3'-dideoxythymidine; D016590:Aphidicolin; D004256:DNA Polymerase I; D004257:DNA Polymerase II; D013936:Thymidine