We examined the activities of human endonuclease III (hNTH1), 8-oxoguanine-DNA glycosylase (hOGG1) and AP endonuclease (hAPE1) in G1 and G2 synchronized human HeLa cells using an oligonucleotide incision assay. Overall the enzymatic activities of hAPE1 and hNTH1 were higher in the G1 compared to the G2 phase of the cell cycle (Figs.
1 and
2). The activity of hOGG1 was much lower and did not vary within the cell cycle (Fig.
3). The low cleavage frequency of substrates containing 8-oxoG was seen even after optimizing experimental conditions. This could be due to low OGG1 expression in these cells. The Western blot analysis of the three repair proteins showed that hNTH1 was expressed at higher levels in G1 phase as compared to G2 phase (Fig.
4). There was no difference in the expression of hAPE1 and OGG1 in G1 and G2 phases. The OGG1 was expressed at very low levels. These results demonstrate the cell cycle phase dependence on the base excision repair of DNA damage. Several studies have analyzed the expression of DNA repair genes throughout the cell cycle. Investigation of the relationship between the cell cycle and
APE1 expression in murine fibroblasts showed that transcription peaks in early S phase [
20]. The expression of hAPE1 is also cell cycle dependent and increases during the G1 phase [
21]. Similar studies with synchronized cells showed that the expression of hNTH1 is regulated during the cell cycle with increased transcription during early and mid S-phase [
22]. The upregulation of other BER proteins in coordination with the S phase has been reported. The expression of
N-alkyl-purine-DNA glycosylase (
MPG) and uracil-DNA glycosylase increases during the G1 phase of the cell cycle, and decreases after mitosis [
21]. Other studies showed that human uracil-DNA glycosylase (
UNG) was increased in late G1/early S phase [
23]. Cell cycle-dependent expression of the human MutY homolog, hMYH, an adenine-specific DNA glycosylase has also been reported [
24]. The levels of hMYH increased during progression of the cell cycle and reached maximum levels in S phase compared to early G1. Similar results were obtained for PCNA [
24].
In contrast, expression levels of hOGG1, TDG DNA glycosylases, O
6
-methylguanine-DNA methyltransferase (MGMT) gene, and the RPA4 genes do not vary with cell cycle [
21]. The OGG1 gene expression and its enzymatic activity in cultured fibroblast cell lines did not vary during the cell cycle [
25]. There were no notable changes in expression of hOGG1 or the human MutT homolog, MTH1, throughout the cell cycle [
24]. It has been shown that OGG1 is a microtubule-associated protein facilitating the movement and redistribution of cytoplasmic OGG1 pools during interphase and mitosis and in response to oxidative DNA damage [
26]. The observed cell cycle dependent differences might reflect distinct roles of individual BER proteins in mutation avoidance. Our data is in agreement with the published literature. Contrary to other reports we did not see alterations in the expression of hAPE1 and such affect could be cell type specific.
Coordinated expression of key DNA repair genes with the cell cycle appears to be a general phenomenon that applies to a number of genes, but it is not universal. The examination of nucleotide excision repair activity (including its coupling to transcription) was found to be similar throughout the cell cycle [
27,
28]. The apparent logic of cell cycle-regulated DNA repair expression (removal of mutagenic and replication-blocking damage ahead of replication forks) requires further exploration. Cell cycle-linked changes in the expression levels of DNA repair genes is one method that cells could use to reduce repair at normal bases and thereby preventing mutations [
29,
30].