Intra- and intercellular variations in the repair efficiency of O6-methylguanine, and their contribution to kinetic...

Following administration to rats of various doses of N-nitrosodimethylamine (NDMA), O(6)-methylguanine (O(6)-meG) was lost from the DNA of four tissues (liver, white blood cells, lymph nodes, bone marrow) over two, sharply demarcated phases with substantially differing repair rates. Repair during each phase followed approximately first-order kinetics in O(6)-meG, even after a high dose of NDMA which caused substantial depletion of O(6)-alkylguanine-DNA alkyltransferase (AGT), a suicide repair protein. This is compatible with rate-determining adduct repair being brought about by a distinct, minor pool of AGT molecules which is rapidly replenished by de novo AGT synthesis. Similar biphasic repair kinetics were also observed in HepG2 cells treated in vitro with NDMA. In this case, the first phase of repair was inhibited by alpha-amanitin, an inhibitor of RNA polymerase II-mediated transcription. However, no dependence on transcriptional activity was found when O(6)-meG repair in specific gene sequences with different transcriptional status in rat liver was examined, suggesting that the effects of alpha-amanitin in HepG2 cells did not reflect inhibition of preferential repair of transcribed sequences. Repair was also examined in rat liver hepatocytes and non-parenchymal cells separately after administration of NDMA at non-AGT depleting doses. Within each cell-population, the repair followed single phase, first-order kinetics, with adduct loss from AGT-rich hepatocytes being significantly faster than from the relatively AGT-deficient non-parenchymal cells. In conclusion, differences in the AGT content of different cell subpopulations in the liver (and probably in other tissues), as well as additional cellular factors affecting repair efficiency, appear to determine the observed variation in the kinetics of repair of O(6)-meG. The additional cellular factors involved appear not to be related to the transcriptional state of the sequences being repaired, but may reflect different states of chromatin condensation.