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… Show more
“…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].…”
Section: Ape1 Is Essential In Mammalian Cellsmentioning
Base excision repair (BER) is an evolutionarily conserved process for maintaining genomic integrity by eliminating several dozen damaged (oxidized or alkylated) or inappropriate bases that are generated endogenously or induced by genotoxicants, predominantly, reactive oxygen species (ROS). BER involves 4-5 steps starting with base excision by a DNA glycosylase, followed by a common pathway usually involving an AP-endonuclease (APE) to generate 3′ OH terminus at the damage site, followed by repair synthesis with a DNA polymerase and nick sealing by a DNA ligase. This pathway is also responsible for repairing DNA single-strand breaks with blocked termini directly generated by ROS. Nearly all glycosylases, far fewer than their substrate lesions particularly for oxidized bases, have broad and overlapping substrate range, and could serve as back-up enzymes in vivo. In contrast, mammalian cells encode only one APE, APE1, unlike two APEs in lower organisms. In spite of overall similarity, BER with distinct subpathways in the mammals is more complex than in E. coli. The glycosylases form complexes with downstream proteins to carry out efficient repair via distinct subpathways one of which, responsible for repair of strand breaks with 3′ phosphate termini generated by the NEIL family glycosylases or by ROS, requires the phosphatase activity of polynucleotide kinase instead of APE1. Different complexes may utilize distinct DNA polymerases and ligases. Mammalian glycosylases have nonconserved extensions at one of the termini, dispensable for enzymatic activity but needed for interaction with other BER and non-BER proteins for complex formation and organelle targeting. The mammalian enzymes are sometimes covalently modified which may affect activity and complex formation. The focus of this review is on the early steps in mammalian BER for oxidized damage.
“…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].…”
Section: Ape1 Is Essential In Mammalian Cellsmentioning
Base excision repair (BER) is an evolutionarily conserved process for maintaining genomic integrity by eliminating several dozen damaged (oxidized or alkylated) or inappropriate bases that are generated endogenously or induced by genotoxicants, predominantly, reactive oxygen species (ROS). BER involves 4-5 steps starting with base excision by a DNA glycosylase, followed by a common pathway usually involving an AP-endonuclease (APE) to generate 3′ OH terminus at the damage site, followed by repair synthesis with a DNA polymerase and nick sealing by a DNA ligase. This pathway is also responsible for repairing DNA single-strand breaks with blocked termini directly generated by ROS. Nearly all glycosylases, far fewer than their substrate lesions particularly for oxidized bases, have broad and overlapping substrate range, and could serve as back-up enzymes in vivo. In contrast, mammalian cells encode only one APE, APE1, unlike two APEs in lower organisms. In spite of overall similarity, BER with distinct subpathways in the mammals is more complex than in E. coli. The glycosylases form complexes with downstream proteins to carry out efficient repair via distinct subpathways one of which, responsible for repair of strand breaks with 3′ phosphate termini generated by the NEIL family glycosylases or by ROS, requires the phosphatase activity of polynucleotide kinase instead of APE1. Different complexes may utilize distinct DNA polymerases and ligases. Mammalian glycosylases have nonconserved extensions at one of the termini, dispensable for enzymatic activity but needed for interaction with other BER and non-BER proteins for complex formation and organelle targeting. The mammalian enzymes are sometimes covalently modified which may affect activity and complex formation. The focus of this review is on the early steps in mammalian BER for oxidized damage.
“…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.…”
Section: Mono-functional and Bi-functional Dna Glycosylases And Ap-simentioning
Base excision repair (BER) proteins act upon a significantly broad spectrum of DNA lesions that result from endogenous and exogenous sources. Multiple sub-pathways of BER (short-path or longpatch) and newly designated DNA repair pathways (e.g., SSBR and NIR) that utilize BER proteins complicate any comprehensive understanding of BER and its role in genome maintenance, chemotherapeutic response, neurodegeneration, cancer or aging. Herein, we propose a unified model of BER, comprised of three functional processes: Lesion Recognition/Strand Scission, Gap Tailoring and DNA Synthesis/Ligation, each represented by one or more multiprotein complexes and coordinated via the XRCC1/DNA Ligase III and PARP1 scaffold proteins. BER therefore may be represented by a series of repair complexes that assemble at the site of the DNA lesion and mediates repair in a coordinated fashion involving protein-protein interactions that dictate subsequent steps or sub-pathway choice. Complex formation is influenced by post-translational protein modifications that arise from the cellular state or the DNA damage response, providing an increase in specificity and efficiency to the BER pathway. In this review, we have summarized the reported BER proteinprotein interactions and protein post-translational modifications and discuss the impact on DNA repair capacity and complex formation.
“…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).…”
Section: Introductionmentioning
confidence: 99%
“…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).…”
Apurinic/apyrimidinic endonuclease 1/redox factor‐1 (APE1/Ref‐1 or APE1) is a multifunctional protein that regulates numerous transcription factors associated with cancer‐related pathways. Because APE1 is essential for cell viability, generation of APE1‐knockout cell lines and determining a comprehensive list of genes regulated by APE1 has not been possible. To circumvent this challenge, we utilized single‐cell RNA sequencing to identify differentially expressed genes (DEGs) in relation to APE1 protein levels within the cell. Using a straightforward yet novel statistical design, we identified 2837 genes whose expression is significantly changed following APE1 knockdown. Using this gene expression profile, we identified multiple new pathways not previously linked to APE1, including the EIF2 signaling and mechanistic target of Rapamycin pathways and a number of mitochondrial‐related pathways. We demonstrate that APE1 has an effect on modifying gene expression up to a threshold of APE1 expression, demonstrating that it is not necessary to completely knockout APE1 in cells to accurately study APE1 function. We validated the findings using a selection of the DEGs along with siRNA knockdown and qRT‐PCR. Testing additional patient‐derived pancreatic cancer cells reveals particular genes (ITGA1,TNFAIP2,COMMD7,RAB3D) that respond to APE1 knockdown similarly across all the cell lines. Furthermore, we verified that the redox function of APE1 was responsible for driving gene expression of mitochondrial genes such as PRDX5 and genes that are important for proliferation such as SIPA1 and RAB3D by treating with APE1 redox‐specific inhibitor, APX3330. Our study identifies several novel genes and pathways affected by APE1, as well as tumor subtype specificity. These findings will allow for hypothesis‐driven approaches to generate combination therapies using, for example, APE1 inhibitor APX3330 with other approved FDA drugs in an innovative manner for pancreatic and other cancer treatments.
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