Activation of the human DNA polymerase β promoter by a DNA-alkylating agent through induced phosphorylation of cAMP response element-binding protein-1

Abstract

Treatment of cells with the DNA-alkylating agent N-methyl-N'-nitro-N- nitrosoguanidine (MNNG) induces expression of the endogenous mammalian DNA polymerase β (β-pol) gene and of the cloned promoter in transient expression studies. The lone cAMP response element (CRE) in the core promoter, along with functional protein kinase A, is critical for the MNNG- induced upregulation. Recently, we described a kinetic mechanism for transcriptional regulation of the β-pol promoter in vitro and found that CRE-binding protein (CREB) from MNNG-treated cells differentially up- regulates the promoter by stimulating formation of closed preinitiation complex (RP(c)). Here, using a CRE-dependent chimeric β-pol promoter, we purified the RP(c) assembled with nuclear extract from MNNG-treated and control HeLa cells. Comparison of proteins in the purified RP(c) samples revealed that the MNNG induction is associated with a strong increase in the Ser133-phosphorylated form of recombinant CREB (CREB-1). CREB depletion of the nuclear extracts diminished transcriptional activity, and addition of purified Ser133-phosphorylated CREB-1 restored activity, whereas unphosphorylated CREB-1 did not. Addition of phosphorylated CREB-1 to the control cell extract mimicked the MNNG-induced up-regulation of transcriptional activity. These results indicate that phosphorylation of CREB-1 is the probable mechanism of activation of the β-pol promoter after treatment of cells with the DNA-alkylating agent MNNG.