DNA damage response of cloned DNA β-polymerase promoter is blocked in mutant cell lines deficient in protein kinase A

Abstract

DNA beta-polymerase (beta-pol), one of the recognized DNA polymerizing enzymes in vertebrates, has a role in 'very short patch' gap-filling synthesis during nucleotide excision DNA repair. In human and mouse, the enzyme is encoded by a single-copy gene located on the short arm of chromosome 8 near the centromere. In a series of studies, we have found that the cloned human beta-pol promoter is regulated by signals acting through the single ATF/CRE palindrome in the core promoter. These signals include transactivation by: adenovirus E1a/E1b proteins; activated p21ras; and in CHO cells, treatment with the DNA damaging agent MNNG. Hence, several types of stimulatory signals are mediated through the single ATF/CRE site, including DNA damage induction. To understand the mechanism of beta-pol promoter activation by MNNG in CHO cells, we asked whether induction of the cAMP/protein kinase A pathway can increase transcription of the cloned promoter in this system. Agents that increase cellular cAMP levels (8-BrcAMP; forskolin and IBMx) activated the beta-pol promoter fusion gene in transient expression experiments, and a mutation in the ATF/CRE palindrome blocked this response. Thus, the ATF/CRE site appears to be cAMP responsive in the CHO cell system. We found that the activation of the cloned beta-pol promoter by MNNG does not occur with two mutant CHO cell lines that are deficient in protein kinase A activity. Further, simultaneous treatment of wild-type CHO cells, with MNNG and to elevate cAMP, failed to result in an additive effect for activation of the beta-pol promoter. Thus, these effectors may act through a common pathway. These results suggest that the activation of the cloned beta-pol promoter in CHO cells following MNNG treatment is mediated through the cAMP/protein kinase A signal transduction pathway.

Activation of the Human DNA Polymerase β Promoter by a DNA-alkylating Agent through Induced Phosphorylation of cAMP Response Element-binding Protein-1

“…Previous studies have shown that the PKA signal transduction pathway is required for MNNG-induced ␤-pol promoter up-regulation (14,15) and that PKA phosphorylates CREB-1 at Ser 133 (23). In the present study, we examined whether the increased CREB-1 phosphorylation after MNNG treatment (Fig.…”

“…This induction required transcription (12), and use of a transfected ␤-pol core promoter fusion gene revealed transcriptional up-regulation of the ␤-pol promoter after MNNG treatment. This response is mediated through the CRE of the ␤-pol promoter (13), and up-regulation of promoter activity is dependent upon the protein kinase A (PKA) signal transduction pathway (14,15).…”

Alternariol induces DNA polymerase β expression through the PKA-CREB signaling pathway

“…In PKA lacking cells MNNG failed to activate the polβ promoter (29). PKA comprises of two catalytic (C) subunits and two regulatory (R) subunits (30).…”

“…The transcriptional activation of polβ promoter is exhibited strongly by DNA damaging agent MNNG treatment of CHO cells (33,34). Mutated polβ promotor fusion genes lacking the element failed to bind protein at this site and failed to respond to MNNG treatment of cells (29). It was shown that the Ser133 phosphorylated CRE binding protein (CREB) was higher versus control in vero cells after 60 min of MNNG treatment (35,36).…”

cAMP response element of murine cytomegalovirus immediate early gene enhancer is transactivated by ras oncogene products

“…Although a ras-dependent phosphorylation of ATF/CREB proteins has not yet been demonstrated, evidence suggesting ATF/CREB responsiveness to p21 r,s has been presented (Kedar et al, 1990;Galien et al, 1991). ATFs/CREBs proteins are a multigene family of transcriptional transactivators that provide trans-criptionally productive interactions when bound to the CREs of gene promoters, and whose binding and/or transcriptional activation functions are modulated by phosphorylations catalysed predominantly by cAMPdependent PKA (Habener, 1990;Englander & Wilson, 1992;de Groot & Sassone-Corsi, 1993;Masson et al, 1993).…”