Analysis of butylphenyl-guanine, butylphenyl-deoxyguanosine, and butylphenyl-deoxyguanosine triphosphate inhibition of DNA replication and ultraviolet-induced DNA repair synthesis using permeable human fibroblasts

Abstract

The purine base and nucleoside analogues N 2-( p- n-butylphenyl-guanine (BuPh-Gua) and N 2-( p- n-butylphenyl)-2′-deoxyguanosine (BuPh-dGuo) are strong inhibitors of isolated mammalian DNA polymerase α, but are less potent than expected as inhibitors of DNA replication in intact cultured cells [G. E. Wright, L. W. Dudycz, Z. Kazimierczuk, N. C. Brown and N. N. Khan, J. med. Chem. 30, 109 (1987)J. The mechanistic basis for these observations was explored using permeable human fibroblasts. DNA replication in the permeable cells was inhibited only slightly by BuPh-Gua and BuPh-dGuo at 100 μM, the highest concentration which could be attained. Similiar results were obtained for ultraviolet-induced DNA repair synthesis, a process which is though to involve the same DNA polymerase as replication. More detailed studies were performed using the corresponding nucleotide analogue, N 2-( p- n-butylphenyl)-2′-deoxyguanosine-5′-triphosphate (BuPh-dGTP), which is much more water-soluble than the base and nucleoside. The apparent K i values for BuPh-dGTP inhibition of both replication and ultraviolet-induced repair synthesis in permeable cells were approximately 3 μM. These values are several hundred-fold greater than the apparent K i for BuPh-dGTP inhibition of isolated human DNA polymerase α, which is approximately 10 nM. We conclude that BuPh-Gua and BuPh-dGuo are poor inhibitors of DNA replication in intact cells not because of permeability barriers, but because, unlike polymerase α, cellular DNA synthesis is relatively insensitive to this group of inhibitors. These results suggest that polymerase α may not be a good general model for predicting the potency of base, deoxyribonucleoside and deoxyribonucleotide analogues as inhibitors of mammalian cellular DNA replication. The fact that the permeable cell systems accurately reflect the relative insensitivity to butylphenyl-guanine derivatives of mammalian DNA replication suggests that permeable cells may be useful tools in future studies of base and nucleoside analogues.