Two divalent metal ions in the active site of a new crystal form of human apurinic/apyrimidinic endonuclease, ape1: implications for the catalytic mechanism 1 1Edited by I. A. Wilson

Beernink, Peter T.; Segelke, Brent W.; Hadi, Masood Z.; Erzberger, J.P.; Wilson, David M.; Rupp, Bernhard

doi:10.1006/jmbi.2001.4529

Cited by 173 publications

(252 citation statements)

References 54 publications

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“…It has previously been proposed that under certain conditions (neutral pH), a second metal binding site (Pb 2+ ) can be observed in APEX1 (16). A comparison of this structure (in the absence of DNA substrates) with our NApe:DNA structures (crystallized at neutral pH), reveals that the proposed second metal ion binding site colocates with the position of the attacking water (Wat3) in NApe.…”

Section: Resultsmentioning

confidence: 54%

Structural basis for the recognition and cleavage of abasic DNA in Neisseria meningitidis

Lü

Šilhán

MacDonald

et al. 2012

Proc. Natl. Acad. Sci. U.S.A.

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Base excision repair (BER) is a highly conserved DNA repair pathway throughout all kingdoms from bacteria to humans. Whereas several enzymes are required to complete the multistep repair process of damaged bases, apurinic-apyrimidic (AP) endonucleases play an essential role in enabling the repair process by recognizing intermediary abasic sites cleaving the phosphodiester backbone 5′ to the abasic site. Despite extensive study, there is no structure of a bacterial AP endonuclease bound to substrate DNA. Furthermore, the structural mechanism for AP-site cleavage is incomplete. Here we report a detailed structural and biochemical study of the AP endonuclease from Neisseria meningitidis that has allowed us to capture structural intermediates providing more complete snapshots of the catalytic mechanism. Our data reveal subtle differences in AP-site recognition and kinetics between the human and bacterial enzymes that may reflect different evolutionary pressures.X-ray crystallography | genome stability | prokaryote A basic (apurinic-apyrimidic, AP) sites within DNA pose a serious threat to all living organisms; if unrepaired, they lead to stalled replication, increased mutations, and an overall loss of genomic integrity. For example human cells defective in AP-site processing are nonviable (1). AP sites occur through either the spontaneous loss of nucleotide bases via chemical breakage of the N-glycosylic bond or as intermediates in the repair of DNA lesions such as oxidatively damaged bases, spontaneously deaminated cytosines, or alkylation of bases (2). It is estimated that 10 4 abasic sites are produced per day per cell in eukaryotes and if left unrepaired will result in significant mutagenic loading on the cell (3, 4). All living organisms have therefore evolved specific enzymes that recognize and repair damaged or missing DNA bases known as the base excision repair (BER) pathway (2).In BER, DNA glycosylases recognize and remove damaged bases to produce abasic sites. The resultant abasic sites are then recognized by AP endonucleases that bind and catalyze a magnesium-dependent 5′ cleavage of the phosphodiester backbone, producing a 3′-OH. Other bifunctional DNA glycosylases cleave the resulting abasic site via an AP lyase mechanism, which leaves a 3′ unsaturated aldehyde (5). In either case, DNA backbone cleavage marks the AP site for subsequent repair by DNA polymerases and ligases. Two main families of AP endonucleases have been identified: the major human AP endonuclease (APEX1) and Escherichia coli homolog ExoIII belong to the ExoIII family of AP endonucleases and are highly conserved (∼51% sequence homology; ∼29% sequence identity). The other is the EndoIV family, named after the E. coli enzyme, and they cleave abasic sites in a Zn 2+ -dependent reaction. Both E. coli and Saccharomyces cerevisiae possess both ExoIII and EndoIV type enzymes, whereas humans have two ExoIII family enzymes.Molecular and biochemical details of abasic DNA recognition and backbone cleavage have mainly focused on the human APEX1 enz...

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Section: Resultsmentioning

confidence: 54%

Structural basis for the recognition and cleavage of abasic DNA in Neisseria meningitidis

Lü

Šilhán

MacDonald

et al. 2012

Proc. Natl. Acad. Sci. U.S.A.

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“…27 and references therein). Notably, this mutant protein exhibits a >56,000,000-fold reduced nuclease capacity, while displaying a better than wild-type AP-DNA binding affinity (Fig.…”

Section: Discussionmentioning

confidence: 99%

“…1A; ref. 27 and references therein). We postulated that simultaneous mutation of these two amino acids would create a catalytically inactive APE1 protein with improved DNA-binding capacity.…”

Section: Ap Endonuclease and Dna Binding Properties Of The Ed Proteinmentioning

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

109

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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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“…The first crystallographic structure of the human APE1 in complex with DNA was obtained using a truncated protein lacking the first 35 amino acids and reveled that this protein consists of two symmetric alpha/beta fold with a significant structural similarity to both bovine DNase I and its E. coli homologue Xth (50). A crystal structure of the full-length protein has also been reported, but again, the Nterminal region was unresolved (10). Three functionally independent domains can be distinguished within the APE1 protein ( Fig.…”

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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Two divalent metal ions in the active site of a new crystal form of human apurinic/apyrimidinic endonuclease, ape1: implications for the catalytic mechanism 1 1Edited by I. A. Wilson

Cited by 173 publications

References 54 publications

Structural basis for the recognition and cleavage of abasic DNA in Neisseria meningitidis

Structural basis for the recognition and cleavage of abasic DNA in Neisseria meningitidis

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

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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