Comparative enzymology of ultraviolet-induced DNA repair synthesis and semiconservative DNA replication in permeable diploid human fibroblasts.

Abstract

In nongrowing mammalian cells, DNA repair synthesis following irradiation with high doses of UV is almost totally inhibited by aphidicolin, an agent specific for DNA polymerase alpha, and presumably is mediated by that polymerase. In this paper, several enzymologic characteristics of DNA repair synthesis induced in permeable confluent diploid human fibroblasts by high doses of UV have been examined and compared with corresponding features of semiconservative DNA replication, a process which is also mediated by polymerase alpha. Inhibition of UV-induced repair synthesis required doses of aphidicolin about 20-fold higher than those needed to inhibit replication, even when the two processes were studied at identical salt and nucleotide concentrations. As is the case for replication, inhibition of UV-induced repair synthesis by aphidicolin is competitive with dCTP. The apparent Ki values for aphidicolin of the two processes are similar, 0.2 microM for repair synthesis and 0.1 microM for semiconservative replication. In contrast, the apparent Km values for dCTP are very different, 0.17 microM for repair synthesis and about 2 microM for replication. The apparent Km values for all four deoxyribonucleoside triphosphates varied together are also very different, 0.07 microM for repair and 30 microM for replication. These results suggest that either UV-induced DNA repair synthesis and semiconservative replication are mediated by two different aphidicolin-sensitive DNA polymerases or the two functions are performed by a single polymerase (e.g. polymerase alpha) which, as a result of accessory proteins or other factors, acquires very different enzymologic characteristics.