Two essential but distinct functions of the mammalian abasic endonuclease

Abstract

The mammalian abasic endonuclease, APE1, has two distinct roles in the repair of oxidative DNA damage and in gene regulation. Here we show that both functions are essential for cell survival. Deletion of the APE1 gene causes embryonic lethality in mice, and no nullizygous embryo fibroblasts have been isolated. We have now established nullizygous embryo fibroblast lines from APE1 ؊/؊ mouse embryos that are transgenic with the ''floxed'' human APE1 (hAPE1) gene. Removal of hAPE1 by Cre expression through nuclear microinjection elicited apoptosis in these cells within 24 h, which was blocked by coinjection of the wild-type hAPE1 gene. In contrast, mutant hAPE1 alleles, lacking either the DNA repair or acetylation-mediated gene regulatory function, could not prevent apoptosis, although the combination of these two mutants complemented APE deficiency induced by Cre. These results indicate that distinct and separable functions of APE1 are both essential for mammalian cells even in vitro and provide the evidence that mammalian cells, unlike yeast or Escherichia coli, absolutely require APE for survival, presumably to protect against spontaneous oxidative DNA damage.conditional gene inactivation ͉ DNA repair ͉ endogenous DNA damage ͉ base excision repair A basic endonuclease (APE), a ubiquitous enzyme, plays a central role in repairing toxic and mutagenic abasic (AP) sites generated in genomes during the repair of oxidation and alkylation damage through the base excision repair (BER) pathway (1). Oxidative DNA lesions, including AP sites, are also spontaneously generated at an estimated rate of 1.5 ϫ 10 5 residues⅐cell Ϫ1 ⅐day Ϫ1 (2). Unlike two distinct APEs present in Escherichia coli and Saccharomyces cerevisiae, only one active APE, APE1, an ortholog of E. coli xth and yeast APN2, has been identified in mammalian cells (3). Based on sequence homology, a second APE-like gene, APE2, was cloned from mammalian cells. However, we could not detect APE activity in the recombinant human APE2 (4), and hAPE2, unlike hAPE1, could not complement yeast APE mutants (5). Although APE-negative bacteria and yeast are viable, very early death (3.5-7.5 days after fertilization) was observed in APE1 nullizygous mouse embryos (6-8). Unlike other BER proteins, e.g., DNA polymerase ␤ and X-ray cross complementation group 1, which are essential for embryonic survival but not for mouse embryonic fibroblasts (MEFs) cultured in vitro (9, 10), APE1-null MEF mutant lines have not been established. The mammalian APE1, independently identified as redox-enhancing factor 1 (Ref1), has a distinct regulatory function in reductively activating C-Jun, p53, and other transcription factors (3, 11) for which Cys-65 (Cys-64 in mouse APE1) was identified as the active site (12). The N-terminal region of the 36-kDa polypeptide, including Cys-65, is not conserved in the E. coli homolog exonuclease III. An additional regulatory function of APE1͞Ref1 was identified in Ca 2ϩ -dependent down-regulation of the parathyroid hormone and renin genes containing negativ...

Early steps in the DNA base excision/single-strand interruption repair pathway in mammalian cells

“…Transgenic expression of Cre in these cells leading to deletion of the APE1 transgene triggered apoptosis. This observation and an independent study provide unequivocal evidence that APE1 is essential even for somatic cells [120,121]. Kelley and his colleagues had earlier shown that APE1 downregulation triggers apoptosis in several human cell lines [122].…”

A unified view of base excision repair: Lesion-dependent protein complexes regulated by post-translational modification

“…The recent demonstration that siRNA may be used to down-regulate APE1 does however show promise for future studies to define the role of APE1 in mammalian cells in vivo [38]. However, complete depletion may be impossible as it is shown to be essential for cellular survival [39]. Although AP-site specific 5'endonucleolytic activity is the major function of APE1, there are a number of minor functions associated with APE1 as well, including 3'phosphodiesterase, 3'phosphoglycolase, 3'phosphatase and 3'-5'exonuclease capabilities.…”

APE1/Ref‐1 knockdown in pancreatic ductal adenocarcinoma – characterizing gene expression changes and identifying novel pathways using single‐cellRNAsequencing

“…Apurinic/apyrimidinic endonuclease 1/redox factor‐1 (APE1/Ref‐1; henceforth referred to as APE1) is a multifunctional protein that is involved in repairing DNA damage via its endonuclease activity in base excision repair (Fung and Demple, 2005; Izumi et al ., 2005; Jiang et al ., 2009; Kelley et al ., 2014), and using its redox protein–protein signaling function to control the activity of numerous transcription factors such as STAT3, NFκB, AP‐1, p53, and hypoxia‐inducible factor 1α (HIF1α), among others (Cardoso et al ., 2012; Fishel et al ., 2015; Gaiddon et al ., 1999; Jiang et al ., 2010; Kelley et al ., 2012; Lando et al ., 2000; Logsdon et al ., 2016). It also contributes to the removal of damaged bases within RNA (Poletto et al ., 2016; Vascotto et al ., 2014).…”

“…Subsequently, it is not possible to generate stable APE1‐knockout cell lines (Tell et al ., 2009). Approaches to circumvent this dilemma have utilized conditional knockouts and siRNA knockdowns (Fung and Demple, 2005; Izumi et al ., 2005; Jiang et al ., 2010). For example, using siRNA knockdowns, our laboratory has previously identified APE1 directly regulating STAT3 transcriptional activity (Cardoso et al ., 2012), suppressing Nrf2‐induced gene expression (Fishel et al ., 2015) and, most recently, regulating carbonic anhydrase 9 (CA9) via HIF‐1α under hypoxic conditions (Logsdon et al ., 2016).…”