Effect of DNA polymerase inhibitors on DNA repair in intact and permeable human fibroblasts: evidence that DNA polymerases delta and beta are involved in DNA repair synthesis induced by N-methyl-N'-nitro-N-nitrosoguanidine.
The involvement of DNA polymerases alpha, beta, and delta in DNA repair synthesis induced by N-methyl-N'-nitro-N-nitrosoguanidine (MNNG) was investigated in human fibroblasts (HF). The effects of anti-(DNA polymerase alpha) monoclonal antibody, (p-n-butylphenyl)deoxyguanosine triphosphate (BuPdGTP), dideoxythymidine triphosphate (ddTTP), and aphidicolin on MNNG-induced DNA repair synthesis were investigated to dissect the roles of the different DNA polymerases. A subcellular system (permeable cells), in which DNA repair synthesis and DNA replication were differentiated by CsCl gradient centrifugation of BrdUMP density-labeled DNA, was used to examine the effects of the polymerase inhibitors. Another approach investigated the effects of several of these inhibitors on MNNG-induced DNA repair synthesis in intact cells by measuring the amount of [3H]thymidine incorporated into repaired DNA as determined by autoradiography and quantitation with an automated video image analysis system. In permeable cells, MNNG-induced DNA repair synthesis was inhibited 56% by 50 micrograms of aphidicolin/mL, 6% by 10 microM BuPdGTP, 13% by anti-(DNA polymerase alpha) monoclonal antibodies, and 29% by ddTTP. In intact cells, MNNG-induced DNA repair synthesis was inhibited 57% by 50 micrograms of aphidicolin/mL and was not significantly inhibited by microinjecting anti-(DNA polymerase alpha) antibodies into HF nuclei. These results indicate that both DNA polymerases delta and beta are involved in repairing DNA damage caused by MNNG.
Hammond
R A
RA
Department of Biochemistry, West Virginia University Health Sciences Center, Morgantown 26506.
McClung
J K
JK
Miller
M R
MR
D000906:Antibodies / Q000008:administration & dosage / Q000494:pharmacology; D016590:Aphidicolin; D002467:Cell Nucleus / Q000378:metabolism; D004256:DNA Polymerase I / Q000037:antagonists & inhibitors* / Q000276:immunology; D004257:DNA Polymerase II / Q000037:antagonists & inhibitors; D004258:DNA Polymerase III; D004260:DNA Repair / Q000187:drug effects*; D004259:DNA-Directed DNA Polymerase / Q000276:immunology; D004224:Diterpenes / Q000494:pharmacology; D004791:Enzyme Inhibitors / Q000494:pharmacology*; D005347:Fibroblasts / Q000378:metabolism*; D006801:Humans; D008769:Methylnitronitrosoguanidine / Q000494:pharmacology; D008845:Microinjections; D019384:Nucleic Acid Synthesis Inhibitors
D000906:Antibodies; D004224:Diterpenes; D004791:Enzyme Inhibitors; D019384:Nucleic Acid Synthesis Inhibitors; D008769:Methylnitronitrosoguanidine; D016590:Aphidicolin; D004256:DNA Polymerase I; D004257:DNA Polymerase II; D004258:DNA Polymerase III; D004259:DNA-Directed DNA Polymerase