APE1 is the major 3'-phosphoglycolate activity in human cell extracts

Parsons, Jason L.; Dianova, Irina I.; Dianov, Grigory L.

doi:10.1093/nar/gkh676

Cited by 120 publications

(116 citation statements)

References 22 publications

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“…The lack of enhanced sensitivity to bleomycin was surprising given that APE1 was found to be the primary activity for excising 3 ¶-phosphoglycolates (a major product of this antibiotic) from single-strand breaks in DNA (47). This observation may suggest that the main toxic intermediates formed by bleomycin are those related to clustered DNA damage, including juxtaposed AP sites and/or 3 ¶ damagecontaining single-strand breaks, or complex DNA doublestrand breaks (48).…”

Section: Discussionmentioning

confidence: 99%

A Dominant-Negative Form of the Major Human Abasic Endonuclease Enhances Cellular Sensitivity to Laboratory and Clinical DNA-Damaging Agents

McNeill

Wilson

2007

Molecular Cancer Research

108

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Apurinic/apyrimidinic (AP) endonuclease 1 (APE1) is the primary enzyme in mammals for the repair of abasic sites in DNA, as well as a variety of 3 ¶ damages that arise upon oxidation or as products of enzymatic processing. If left unrepaired, APE1 substrates can promote mutagenic and cytotoxic outcomes. We describe herein a dominant-negative form of APE1 that lacks detectable nuclease activity and binds substrate DNA with a 13-fold higher affinity than the wild-type protein. This mutant form of APE1, termed ED, possesses two amino acid substitutions at active site residues Glu 96 (changed to Gln) and Asp 210 (changed to Asn). In vitro biochemical assays reveal that ED impedes wild-type APE1 AP site incision function, presumably by binding AP-DNA and blocking normal lesion processing. Moreover, tetracycline-regulated (tet-on) expression of ED in Chinese hamster ovary cells enhances the cytotoxic effects of the laboratory DNA-damaging agents, methyl methanesulfonate (MMS; 5.4-fold) and hydrogen peroxide (1.5-fold). This MMS-induced, ED-dependent cell killing coincides with a hyperaccumulation of AP sites, implying that excessive DNA damage is the cause of cell death. Because an objective of the study was to identify a protein reagent that could be used in targeted gene therapy protocols, the effects of ED on cellular sensitivity to a number of chemotherapeutic compounds was tested. We show herein that ED expression sensitizes Chinese hamster ovary cells to the killing effects of the alkylating agent 1,3-bis(2-chloroethyl)-1-nitrosourea (also known as carmustine) and the chain terminating nucleoside analogue dideoxycytidine (also known as zalcitabine), but not to the radiomimetic bleomycin, the nucleoside analogue B-D-arabinofuranosylcytosine (also known as cytarabine), the topoisomerase inhibitors camptothecin and etoposide, or the cross-linking agents mitomycin C and cisplatin. Transient expression of ED in the human cancer cell line NCI-H1299 enhanced cellular sensitivity to MMS, 1,3-bis(2-chloroethyl)-1-nitrosourea, and dideoxycytidine, demonstrating the potential usefulness of this strategy in the treatment of human tumors.

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Section: Discussionmentioning

confidence: 99%

A Dominant-Negative Form of the Major Human Abasic Endonuclease Enhances Cellular Sensitivity to Laboratory and Clinical DNA-Damaging Agents

McNeill

Wilson

2007

Molecular Cancer Research

108

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“…Such non-conventional groups arise as products of hydroxyl radical attack of the sugar phosphate backbone or as repair intermediates during DNA processing. Experiments using human whole cell extracts have shown that APE1 is rate limiting for the repair of DNA strand breaks induced by hydrogen peroxide (presumably 3¢-phosphates) and bleomycin (3¢-phosphoglycolates) (89), and is the predominant enzyme for the excision of 3¢-phosphoglycolate residues from a single nucleotide gap (160). Biochemical studies also indicate that APE1 plays a prominent role in the removal of 3¢-deoxyribose fragments that are generated by AP lyase b-elimination reactions, which are catalyzed by certain multi-functional DNA glycosylases (e.g., 8-oxoguanine DNA glycosylase [OGG1] and endonuclease III homolog 1 [NTH1]) or are promoted ''non-specifically'' by basic polypeptides, such as polyamines or histones [reviewed in Doetsch and Cunningham (41) and Hegde et al (75)].…”

Section: ¢-Repair Diesterasementioning

confidence: 99%

Human Apurinic/Apyrimidinic Endonuclease 1

2014

Antioxidants & Redox Signaling

216

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Significance: Human apurinic/apyrimidinic endonuclease 1 (APE1, also known as REF-1) was isolated based on its ability to cleave at AP sites in DNA or activate the DNA binding activity of certain transcription factors. We review herein topics related to this multi-functional DNA repair and stress-response protein. Recent Advances: APE1 displays homology to Escherichia coli exonuclease III and is a member of the divalent metal-dependent a/b fold-containing phosphoesterase superfamily of enzymes. APE1 has acquired distinct active site and loop elements that dictate substrate selectivity, and a unique N-terminus which at minimum imparts nuclear targeting and interaction specificity. Additional activities ascribed to APE1 include 3¢-5¢ exonuclease, 3¢-repair diesterase, nucleotide incision repair, damaged or site-specific RNA cleavage, and multiple transcription regulatory roles. Critical Issues: APE1 is essential for mouse embryogenesis and contributes to cell viability in a genetic background-dependent manner. Haploinsufficient APE1 +/ -mice exhibit reduced survival, increased cancer formation, and cellular/tissue hyper-sensitivity to oxidative stress, supporting the notion that impaired APE1 function associates with disease susceptibility. Although abnormal APE1 expression/localization has been seen in cancer and neuropathologies, and impaired-function variants have been described, a causal link between an APE1 defect and human disease remains elusive. Future Directions: Ongoing efforts aim at delineating the biological role(s) of the different APE1 activities, as well as the regulatory mechanisms for its intra-cellular distribution and participation in diverse molecular pathways. The determination of whether APE1 defects contribute to human disease, particularly pathologies that involve oxidative stress, and whether APE1 small-molecule regulators have clinical utility, is central to future investigations. Antioxid. Redox Signal. 20,

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“…Untagged full-length human TTRAP yeast expression vectors were created by cloning the appropriate TTRAP ORF from pCRII-TOPO (NdeI-BamHI) into NheIBamHI of pVTU260 (Euroscarf, kindly provided by Dr. Eva Hoffmann). pET16b-APE1 has been described previously 28 , and APE1 ORF was cloned into BamHI-EcoRI sites of pAS2-1 (Clontech). pEGFP-TTRAP was created by cloning a BamHI-EcoRI fragment of pACT-TTRAP-2 into BglII-EcoRI of pEGFP-C3 (Clontech).…”

Section: Dna Constructsmentioning

confidence: 99%

TDP2 protects transcription from abortive topoisomerase activity and is required for normal neural function

et al. 2014

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Here, we identify such an enzyme in human cells and show that this activity efficiently restores 5'-phosphate termini at DNA double-strand breaks in preparation for DNA ligation. We reveal that this enzyme is TTRAP, a member of the Mg 2+ /Mn 2+ -dependent family of phosphodiesterases, and show that cellular depletion of TTRAP results in increased susceptibility and sensitivity to topoisomerase II-induced DNA double-strand breaks. TTRAP is the first human 5'-tyrosine phosphodiesterase to be identified and we suggest this enzyme additionally be denoted tyrosyl DNA phosphodiesterase-2 (TDP2).In an attempt to identify novel human tyrosyl DNA phosphodiesterase activities, we exploited the hypersensitivity of Saccharomyces cerevisiae tdp1Δ rad1Δ double mutant cells to camptothecin (CPT), a topoisomerase I (Top1) poison that induces single-strand breaks (SSBs) with Top1 covalently linked to the 3'-terminus 3, 10 . This strain lacks not only Tdp1 but also Rad1-Rad10 nuclease, which in yeast provides an alternative (endonucleolytic) pathway for removing Top1 from 3'-termini 11,12 . We transformed this strain with a human cDNA library 3 and screened the resulting population of transformants for cellular resistance to CPT. Of six tdp1Δ rad1Δ transformants displaying wild-type levels of CPT resistance, three harboured cDNA clones encoding TDP1 and three harboured cDNA clones encoding TTRAP (TRAF and TNF receptor-associated protein), a protein of unknown function and a putative member of the Mg 2+ /Mn 2+ -dependent phosphodiesterase super-family, with the DNA repair protein AP endonuclease-1 (APE1) being its closest relative 13,14 (Fig.1a and Supplementary Fig.1).The TDP1 and TTRAP cDNA clones recovered in the genetic screen suppressed the CPT sensitivity of tdp1Δ rad1Δ cells to a similar extent (Fig. 1b & data not shown). Whilst the pACT-TTRAP clones encoded TTRAP protein that lacked eight (pACT-TTRAP-2, pACT-TTRAP-3) or twenty-two (pACT-TTRAP-1) residues from the amino-terminus (data not shown), full-length TTRAP similarly suppressed the CPT sensitivity of tdp1Δ rad1Δ (Fig. 1c, left). In contrast, human APE1 protein failed to suppress this sensitivity, suggesting that ability to complement CPT sensitivity in tdp1Δ rad1Δ cells is not a generic feature of metaldependent phosphodiesterases (Fig. 1c, left). Conversely, whereas human APE1 suppressed the sensitivity of AP endonuclease-defective apn1Δapn2Δ tpp1Δ yeast cells to methyl methanesulphonate (MMS)-induced DNA base damage, human TTRAP did not, suggesting that the impact of TTRAP in these experiments was restricted to topoisomerase-mediated DNA damage ( Fig. 1c, right). TTRAP contains four highly conserved motifs that putatively assign this protein to the metal-dependent phosphodiesterase superfamily (see Fig.1a and Supplementary Fig.1). We thus examined whether mutation of two predicted catalytic residues (Fig.1a; E152 and D262) within two of these motifs impacted on the complementation of CPT sensitivity by TTRAP. Indeed, in contrast to wild-type TTRAP protein, ne...

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APE1 is the major 3'-phosphoglycolate activity in human cell extracts

Cited by 120 publications

References 22 publications

A Dominant-Negative Form of the Major Human Abasic Endonuclease Enhances Cellular Sensitivity to Laboratory and Clinical DNA-Damaging Agents

A Dominant-Negative Form of the Major Human Abasic Endonuclease Enhances Cellular Sensitivity to Laboratory and Clinical DNA-Damaging Agents

Human Apurinic/Apyrimidinic Endonuclease 1

TDP2 protects transcription from abortive topoisomerase activity and is required for normal neural function

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