Treatment of hamster cells in culture with the DNA alkylating agent N-methyl-N'-nitro-N-nitrosoguanidine (MNNG) induces DNA polymerase beta (beta-pol) gene expression and cellular levels of the enzyme. Transcriptional activity of a cloned beta-pol promoter in transient expression assays is also stimulated. Among the requirements for these responses are methylation damage to genomic DNA, cellular cAMP-dependent protein kinase, and the ATF/CREB site of the cloned beta-pol promoter. In the present study, HeLa cell nuclear extract from MNNG-treated cells was much more active in an in vitro transcription assay than nuclear extract from normal cells. By using an oligonucleotide affinity column to deplete the nuclear extract of ATF/CREB, we showed that the difference was due to ATF/CREB activator. Purified ATF/CREB activator from MNNG-treated cells was approximately 10-fold more active than ATF/CREB purified from normal cells as a transcriptional activator for the depleted nuclear extract. ATF/CREB in the extract from normal cells is known to activate in vitro transcription by increasing the rate of promoter clearance [Narayan, S., Widen, S. G., Beard, W. A., & Wilson, S. H. (1994) J. Biol. Chem. 269, 12755-12763]. With ATF/CREB from MNNG-treated cells, the amount of preinitiation complex formed was much greater than with ATF/CREB from normal cells, and the kinetics of both the closed to open preinitiation complex isomerization and promoter clearance were altered. These results indicate that the mechanism of transcriptional activation secondary to DNA alkylation damage is recruitment of more preinitiation complex and alteration of the kinetic scheme of transcription initiation.