Characterization of the promoter region of the human apurinic endonuclease gene (APE).
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.
Harrison
L
L
Department of Molecular and Cellular Toxicology, Harvard School of Public Health, Boston, Massachusetts 02115.
Ascione
A G
AG
Wilson
D M
DM
Demple
B
B
D000818:Animals; D001483:Base Sequence; D006528:Carcinoma, Hepatocellular; D002460:Cell Line; D015500:Chloramphenicol O-Acetyltransferase / Q000096:biosynthesis; D017931:DNA Primers; D015342:DNA Probes; D043603:DNA-(Apurinic or Apyrimidinic Site) Lyase; D043223:Deoxyribonuclease IV (Phage T4-Induced); D017353:Gene Deletion; D015870:Gene Expression; D006367:HeLa Cells; D015186:Hominidae / Q000235:genetics*; D006801:Humans; D015464:Leukemia, Myelogenous, Chronic, BCR-ABL Positive; D008113:Liver Neoplasms; D008190:Lyases / Q000096:biosynthesis* / Q000235:genetics*; D008969:Molecular Sequence Data; D009838:Oligodeoxyribonucleotides; D011401:Promoter Regions, Genetic; D015183:Restriction Mapping; D013755:Tetradecanoylphorbol Acetate / Q000494:pharmacology; D014158:Transcription, Genetic; D014162:Transfection; D014407:Tumor Cells, Cultured
D017931:DNA Primers; D015342:DNA Probes; D009838:Oligodeoxyribonucleotides; D015500:Chloramphenicol O-Acetyltransferase; D043223:Deoxyribonuclease IV (Phage T4-Induced); D008190:Lyases; D043603:DNA-(Apurinic or Apyrimidinic Site) Lyase; D013755:Tetradecanoylphorbol Acetate