Human Apurinic/Apyrimidinic Endonuclease (Ape1) and Its N-terminal Truncated Form (AN34) Are Involved in DNA Fragmentation during Apoptosis

Yoshida, Akira; Urasaki, Yoshimasa; Waltham, Mark; Bergman, Ann Charlotte; Pourquier, Philippe; Rothwell, Dominic G.; Inuzuka, Manabu; Weinstein, John N.; Ueda, Takanori; Appella, Ettore; Hickson, Ian D.; Pommier, ﻿Yves

doi:10.1074/jbc.m304914200

Cited by 52 publications

(45 citation statements)

References 47 publications

(49 reference statements)

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“…Mitochondrial localization of APE1 could be associated with a potential role in mtDNA repair of oxidized bases (11,33,54). However, since it is not clear whether N⌬33APE1 maintains its DNA repair activity in vivo (16) or acquires an aspecific endonuclease activity for double-stranded DNA (dsDNA) (66), at present it is not possible to drive any definitive conclusion. Moreover, as the truncated N⌬33APE1 form is associated with an apoptotic phenotype (23), it cannot be excluded that its generation may causatively be involved in the cytotoxic effect, driving proapoptotic triggering directly within mitochondria.…”

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confidence: 84%

APE1/Ref-1 Interacts with NPM1 within Nucleoli and Plays a Role in the rRNA Quality Control Process

Vascotto

Fantini

Romanello

et al. 2009

Molecular and Cellular Biology

220

411

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APE1/Ref-1 (hereafter, APE1), a DNA repair enzyme and a transcriptional coactivator, is a vital protein in mammals. Its role in controlling cell growth and the molecular mechanisms that fine-tune its different cellular functions are still not known. By an unbiased proteomic approach, we have identified and characterized several novel APE1 partners which, unexpectedly, include a number of proteins involved in ribosome biogenesis and RNA processing. In particular, a novel interaction between nucleophosmin (NPM1) and APE1 was characterized. We observed that the 33 N-terminal residues of APE1 are required for stable interaction with the NPM1 oligomerization domain. As a consequence of the interaction with NPM1 and RNA, APE1 is localized within the nucleolus and this localization depends on cell cycle and active rRNA transcription. NPM1 stimulates APE1 endonuclease activity on abasic double-stranded DNA (dsDNA) but decreases APE1 endonuclease activity on abasic single-stranded RNA (ssRNA) by masking the N-terminal region of APE1 required for stable RNA binding. In APE1-knocked-down cells, pre-rRNA synthesis and rRNA processing were not affected but inability to remove 8-hydroxyguanine-containing rRNA upon oxidative stress, impaired translation, lower intracellular protein content, and decreased cell growth rate were found. Our data demonstrate that APE1 affects cell growth by directly acting on RNA quality control mechanisms, thus affecting gene expression through posttranscriptional mechanisms.APE1/Ref-1 (also called HAP1 or APEX, and here referred to as APE1), the mammalian ortholog of Escherichia coli Xth (exonuclease III), is a vital protein (20) that acts as a master regulator of cellular response to oxidative stress conditions and contributes to the maintenance of genome stability (55, 56). APE1 is involved in both the base excision repair (BER) pathways of DNA lesions, acting as the major apurinic/apyrimidinic (AP) endonuclease, and in transcriptional regulation of gene expression as a redox coactivator of different transcription factors, such as early growth response protein 1 (Egr-1), NF-B, and p53 (55, 56). These two biological activities are located in two functionally distinct protein domains. In fact, the N-terminal region, containing the nuclear localization signal (NLS) sequence, is principally devoted to redox activity through Cys65, while the C-terminal one exerts enzymatic activity on the abasic sites of DNA (56, 63). The protein C terminus is highly conserved during phylogenesis, while the N terminus is not. Except in mammals, which always show a high sequence conservation (more than 90%), and Danio, Drosophila, Xenopus, and Dictyostelium (presenting a sequence identity of less than 40%), the N-terminal region is mostly absent in other organisms. A third APE1 function, which is regulated by Lys6/Lys7 acetylation (7), is indirect binding to the negative calcium response elements (nCaRE) of some promoters (i.e., PTH and APE1 promoters), thus acting as a transcriptional repressor (12,30).In different ...

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confidence: 84%

APE1/Ref-1 Interacts with NPM1 within Nucleoli and Plays a Role in the rRNA Quality Control Process

Vascotto

Fantini

Romanello

et al. 2009

Molecular and Cellular Biology

220

411

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“…Gel images were generated using a FluorChem 8900 imaging system (Alpha Innotech) and scanned using ImageQuant TL version7.0 to obtain densitometric profiles and to determine the final TREX1 protein concentrations. The NM23-H1 (26) and APE1 fragment (amino acids 32-318) (27) endonucleases were cloned into plasmids as N-terminal MBP fusions with a PreScission protease recognition sequence between the MBP and the endonuclease. The MBP fusion endonucleases were overexpressed in Escherichia coli BL21(DE3) Rosetta 2 cells (Novagen), bound to amylose resins (New England Biolabs), and washed, and the column resin was incubated at 4°C overnight with PreScission protease (GE Biosciences) to separate the endonuclease from MBP.…”

Section: Methodsmentioning

confidence: 99%

Dominant Mutations of the TREX1 Exonuclease Gene in Lupus and Aicardi-Goutières Syndrome

Fye

Orebaugh

Coffin

et al. 2011

Journal of Biological Chemistry

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TREX1 is a potent 335 exonuclease that degrades singleand double-stranded DNA (ssDNA and dsDNA). TREX1 mutations at amino acid positions Asp-18 and Asp-200 in familial chilblain lupus and Aicardi-Goutières syndrome elicit dominant immune dysfunction phenotypes. Failure to appropriately disassemble genomic DNA during normal cell death processes could lead to persistent DNA signals that trigger the innate immune response and autoimmunity. We tested this concept using dsDNA plasmid and chromatin and show that the TREX1 exonuclease locates 3 termini generated by endonucleases and degrades the nicked DNA polynucleotide. A competition assay was designed using TREX1 dominant mutants and variants to demonstrate that an intact DNA binding process, coupled with dysfunctional chemistry in the active sites, explains the dominant phenotypes in TREX1 D18N, D200N, and D200H alleles. The TREX1 residues Arg-174 and Lys-175 positioned adjacent to the active sites act with the Arg-128 residues positioned in the catalytic cores to facilitate melting of dsDNA and generate ssDNA for entry into the active sites. Metal-dependent ssDNA binding in the active sites of the catalytically inactive dominant TREX1 mutants contributes to DNA retention and precludes access to DNA 3 termini by active TREX1 enzyme. Thus, the dominant disease genetics exhibited by the TREX1 D18N, D200N, and D200H alleles parallel precisely the biochemical properties of these TREX1 dimers during dsDNA degradation of plasmid and chromatin DNA in vitro. These results support the concept that failure to degrade genomic dsDNA is a principal pathway of immune activation in TREX1-mediated autoimmune disease.

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“…Three functionally independent domains can be distinguished within the APE1 protein ( Fig. 5): (i) the first 33-35 amino acid region consists of a structurally disordered segment (133) essential for the interaction with other proteins (148,149) and harboring sites for PTMs (14,24,70,89,159,165,166) and RNA interaction (89); (ii) the redox domain is located in a region between amino acids 35 and 127; and (iii) the DNA repair domain, which spans the C-terminal domain of the protein from about residue 61 onwards (42,68). Whereas the APE1 C-terminal domain involved in AP endonuclease activity is conserved from bacteria to humans, the N-terminal region is unique to mammals suggesting a recent acquisition during evolution.…”

Section: Fig 5 Schematic Representationmentioning

confidence: 99%

Emerging Roles of the Nucleolus in Regulating the DNA Damage Response: The Noncanonical DNA Repair Enzyme APE1/Ref-1 as a Paradigmatical Example

Antoniali

Lirussi

Poletto

et al. 2014

Antioxidants & Redox Signaling

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Significance: An emerging concept in DNA repair mechanisms is the evidence that some key enzymes, besides their role in the maintenance of genome stability, display also unexpected noncanonical functions associated with RNA metabolism in specific subcellular districts (e.g., nucleoli). During the evolution of these key enzymes, the acquisition of unfolded domains significantly amplified the possibility to interact with different partners and substrates, possibly explaining their phylogenetic gain of functions. Recent Advances: After nucleolar stress or DNA damage, many DNA repair proteins can freely relocalize from nucleoli to the nucleoplasm. This process may represent a surveillance mechanism to monitor the synthesis and correct assembly of ribosomal units affecting cell cycle progression or inducing p53-mediated apoptosis or senescence. Critical Issues: A paradigm for this kind of regulation is represented by some enzymes of the DNA base excision repair (BER) pathway, such as apurinic/apyrimidinic endonuclease 1 (APE1). In this review, the role of the nucleolus and the noncanonical functions of the APE1 protein are discussed in light of their possible implications in human pathologies. Future Directions: A productive cross-talk between DNA repair enzymes and proteins involved in RNA metabolism seems reasonable as the nucleolus is emerging as a dynamic functional hub that coordinates cell growth arrest and DNA repair mechanisms. These findings will drive further analyses on other BER proteins and might imply that nucleic acid processing enzymes are more versatile than originally thought having evolved DNA-targeted functions after a previous life in the early RNA world. Antioxid. Redox Signal. 20, 621-639.

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Human Apurinic/Apyrimidinic Endonuclease (Ape1) and Its N-terminal Truncated Form (AN34) Are Involved in DNA Fragmentation during Apoptosis

Cited by 52 publications

References 47 publications

APE1/Ref-1 Interacts with NPM1 within Nucleoli and Plays a Role in the rRNA Quality Control Process

APE1/Ref-1 Interacts with NPM1 within Nucleoli and Plays a Role in the rRNA Quality Control Process

Dominant Mutations of the TREX1 Exonuclease Gene in Lupus and Aicardi-Goutières Syndrome

Emerging Roles of the Nucleolus in Regulating the DNA Damage Response: The Noncanonical DNA Repair Enzyme APE1/Ref-1 as a Paradigmatical Example

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