Abstract
Apurinic/apyrimidinic (AP) sites are mutagenic and block DNA synthesis in vitro. Repair of AP sites is initiated by AP endonucleases that cleave just 5' to the damage. We linked a 4.1-kilobase pair HindIII DNA fragment from the region upstream of the human AP endonuclease gene (APE) to the chloramphenicol acetyltransferase (CAT) gene. Deletions generated constructs containing 1.9 kilobase pairs to 50 base pairs (bp) of the APE upstream region. Transient transfection studies in HeLa cells established that the basal APE promoter is contained within a 500-bp fragment. The major transcriptional start site in HeLa, hepatoma (HepG2), and myeloid leukemic (K562) cells was mapped to a cluster of sites approximately 130 bp downstream of a putative "CCAAT box," approximately 130 bp 5' of the first splice junction in APE. Deletion of 5' sequences to within 10 bp of the CCAAT box reduced the CAT activity by only about half, and removal of the CCAAT box region left a residual promotor activity approximately 9%. Deletion to 31 bp upstream of the transcriptional start site abolished APE promoter activity. DNA sequence analysis revealed potential transcription factor recognition sites in the APE promoter. Gel mobility-shift assays showed that both human upstream factor and Sp1 can bind their respective sites in the APE promoter. However, DNase I footprinting using HeLa nuclear extract showed that the binding of Sp1 and upstream factor is blocked by the binding of other proteins to the nearby CCAAT box region.
Highlights
Apurinic endonucleases initiate the repair of apuriniclapyrimidinic (AP)! sites, of which perhaps thousands per day are introduced into the human genome by spontaneous base hydrolysis and reactions with oxygen radicals and other cellular metabolites (Lindahl, 1993)
Our own recent work indicates possible transcriptional regulation of AP endonuclease gene (APE) during epithelial wound healing," and the recently reported hypoxic response of APE (Yao et al, 1994) shows that APE transcription is regulated under some circumstances
We analyzed the functional elements of the APE promoter
Summary
Apurinic endonucleases initiate the repair of apuriniclapyrimidinic (AP)! sites, of which perhaps thousands per day are introduced into the human genome by spontaneous base hydrolysis and reactions with oxygen radicals and other cellular metabolites (Lindahl, 1993). Molecular studies of prokaryotic and eukaryotic AP endonucleases have revealed two families of proteins: those related to Escherichia coli endonuclease IV and yeast Apnl, or those related to E. coli exonuclease III and the major human AP endonuclease, Ape (Demple and Harrison, 1994). Both enzyme families are "class II" AP endonucleases, which hydrolyze the phosphodiester bond on the 5' of AP sites (Levin and Demple, 1990) to allow excision and repair DNA synthesis (Demple and Harrison, 1994). The work presented here identifies promoter elements and DNA-binding activities that mediate basal APE expression in cultured cells
Sign-in/Register to view highlights & summary Sign-in/Register to access full text options 
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a callDisclaimer: All third-party content on this website/platform is and will remain the property of their respective owners and is provided on "as is" basis without any warranties, express or implied. Use of third-party content does not indicate any affiliation, sponsorship with or endorsement by them. Any references to third-party content is to identify the corresponding services and shall be considered fair use under The CopyrightLaw.
