Abstract
Abasic (apurinic/apyrimidinic or AP) sites are a frequent type of DNA damage that threatens genetic stability. The predominant mammalian enzyme initiating repair of AP sites is the Ape1 AP endonuclease (also called Apex or Hap1), which also facilitates DNA binding by several transcription factors (Ref1 activity). We found that expression of the APE1 gene was coordinated with the cell cycle in murine NIH3T3 cells: APE1 mRNA levels rose after the G(1)-S transition and peaked approximately 4-fold higher in early to mid-S phase. The increased APE1 mRNA was the result of transcriptional activation rather than increased mRNA stability. Fusions of various APE1 promoter fragments to the chloramphenicol acetyltransferase CAT reporter gene indicated that APE1 expression depends on two transcription factor Sp1 binding sites within the promoter region. Mutation of these sites or of two CCAAT elements within the APE1 promoter, in conjunction with protein binding studies, demonstrated their specific roles. The Sp1 site upstream of the transcription start, together with an adjacent CCAAT element, establishes a protein-DNA complex required for basal transcription of APE1. The Sp1 site downstream of the transcription start was required for the response to cell growth. Because Ape1 is a dual function enzyme, its cell cycle-dependent expression might affect both DNA repair and the activity of various transcription factors as a function of the cell cycle.
Highlights
Mutagenesis and cell proliferation are essential steps leading to carcinogenesis
We found that expression of the APE1 gene was coordinated with the cell cycle in murine NIH3T3 cells: APE1 mRNA levels rose after the G1-S transition and peaked ϳ4-fold higher in early to mid-S phase
Because transcription factors E2F and Sp1 have been implicated as central mediators of transcriptional control during progression into S phase [29], we examined their roles in cell cycle-dependent APE1 expression
Summary
Cell Culture—Murine NIH3T3 embryonic fibroblast cells (obtained from ATCC) were grown in Dulbecco’s modified Eagle’s medium supplemented with 10% (v/v) bovine serum (Hyclone) under 5% (v/v) CO2 at 37 °C. Cells were plated at 10% confluence in normal growth medium with 10% bovine serum overnight. The cultures were shifted to starvation medium (0.5% bovine serum) for 48 – 60 h. Complete medium containing 10% bovine serum was added. Flow Cytometric Analysis—To monitor the progression of cells through the cell cycle, DNA content was measured by flow cytometric
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