CHARACTERIZATION OF THE PROMOTER REGION OF THE HUMAN APURINIC ENDONUCLEASE GENE (APE)

被引:38
作者
HARRISON, L [1 ]
ASCIONE, AG [1 ]
WILSON, DM [1 ]
DEMPLE, B [1 ]
机构
[1] HARVARD UNIV,SCH PUBL HLTH,DEPT MOLEC & CELLULAR TOXICOL,BOSTON,MA 02115
关键词
D O I
10.1074/jbc.270.10.5556
中图分类号
Q5 [生物化学]; Q7 [分子生物学];
学科分类号
071010 ; 081704 ;
摘要
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 similar to 130 bp downstream of a putative ''CCAAT box,'' similar to 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 promoter activity similar to 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 Spl 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.
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收藏
页码:5556 / 5564
页数:9
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