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PDBsum entry 1e9n

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protein metals Protein-protein interface(s) links
DNA repair PDB id
1e9n
Jmol
Contents
Protein chain
274 a.a. *
Metals
_PB ×4
Waters ×349
* Residue conservation analysis
PDB id:
1e9n
Name: DNA repair
Title: A second divalent metal ion in the active site of a new crystal form of human apurinic/apyrimidinic endonuclease, ape1, and its implications for the catalytic mechanism
Structure: DNA-(apurinic or apyrimidinic site) lyase. Chain: a, b. Synonym: ap endonuclease 1, hap1, ref1, ape1. Engineered: yes
Source: Homo sapiens. Human. Organism_taxid: 9606. Gene: ape1. Expressed in: escherichia coli. Expression_system_taxid: 469008.
Resolution:
2.2Å     R-factor:   0.186     R-free:   0.252
Authors: P.T.Beernink,B.W.Segelke,B.Rupp
Key ref:
P.T.Beernink et al. (2001). Two divalent metal ions in the active site of a new crystal form of human apurinic/apyrimidinic endonuclease, Ape1: implications for the catalytic mechanism. J Mol Biol, 307, 1023-1034. PubMed id: 11286553 DOI: 10.1006/jmbi.2001.4529
Date:
24-Oct-00     Release date:   16-Feb-01    
PROCHECK
Go to PROCHECK summary
 Headers
 References

Protein chains
Pfam   ArchSchema ?
P27695  (APEX1_HUMAN) -  DNA-(apurinic or apyrimidinic site) lyase
Seq:
Struc:
318 a.a.
274 a.a.
Key:    PfamA domain  PfamB domain  Secondary structure  CATH domain

 Enzyme reactions 
   Enzyme class: E.C.4.2.99.18  - DNA-(apurinic or apyrimidinic site) lyase.
[IntEnz]   [ExPASy]   [KEGG]   [BRENDA]
      Reaction: The C-O-P bond 3' to the apurinic or apyrimidinic site in DNA is broken by a beta-elimination reaction, leaving a 3'-terminal unsaturated sugar and a product with a terminal 5'-phosphate.
 Gene Ontology (GO) functional annotation 
  GO annot!
  Cellular component     intracellular   12 terms 
  Biological process     cellular response to cAMP   24 terms 
  Biochemical function     protein binding     27 terms  

 

 
DOI no: 10.1006/jmbi.2001.4529 J Mol Biol 307:1023-1034 (2001)
PubMed id: 11286553  
 
 
Two divalent metal ions in the active site of a new crystal form of human apurinic/apyrimidinic endonuclease, Ape1: implications for the catalytic mechanism.
P.T.Beernink, B.W.Segelke, M.Z.Hadi, J.P.Erzberger, D.M.Wilson, B.Rupp.
 
  ABSTRACT  
 
The major human abasic endonuclease, Ape1, is an essential DNA repair enzyme that initiates the removal of apurinic/apyrimidinic sites from DNA, excises 3' replication-blocking moieties, and modulates the DNA binding activity of several transcriptional regulators. We have determined the X-ray structure of the full-length human Ape1 enzyme in two new crystal forms, one at neutral and one at acidic pH. The new structures are generally similar to the previously determined structure of a truncated Ape1 protein, but differ in the conformation of several loop regions and in spans of residues with weak electron density. While only one active-site metal ion is present in the structure determined at low pH, the structure determined from a crystal grown at the pH optimum of Ape1 nuclease activity, pH 7.5, has two metal ions bound 5 A apart in the active site. Enzyme kinetic data indicate that at least two metal-binding sites are functionally important, since Ca(2+) exhibits complex stimulatory and inhibitory effects on the Mg(2+)-dependent catalysis of Ape1, even though Ca(2+) itself does not serve as a cofactor. In conjunction, the structural and kinetic data suggest that Ape1 catalyzes hydrolysis of the DNA backbone through a two metal ion-mediated mechanism.
 
  Selected figure(s)  
 
Figure 3.
Figure 3. Active site region of human Ape1 crystal form III. (a) wARP electron density map (see Materials and Methods) showing the two Pb ions (green spheres) bound at the active site. The map is shown at contour levels of 1.5s (blue) and 5s (red). The image was generated with XtalView [32] and Raster3D [33]. (b) Stick rendering of the same region, showing electrostatic interactions. The two Pb ions are shown as pink spheres and water molecules are shown as red spheres. The interactions (non-hydrogen atoms <3.3 Å apart) are depicted as dotted lines; metal interactions (magenta), water interactions (red) and protein interactions (black) are shown. The image was generated with InsightII (BIOSYM/Molecular Simulations, Inc., San Diego, CA).
Figure 5.
Figure 5. Proposed catalytic mechanism for Ape1 hydrolysis of DNA. (a) A portion of the DNA backbone is shown at the top, with the abasic residue shown on the 5' (right) side. The continuation of the DNA backbone is represented by R groups and wavy lines. The primary residues coordinating the two metal ions are shown, with site A (Asp70 and Glu96) on the left and site B (Asp210, Asn212 and His309) on the right. In the proposed mechanism, the Mg2+ in site A coordinates an hydroxyl ion, which carries out nucleophilic attack on the phosphorous 5' to the abasic nucleotide. The Mg2+ in site B acts to neutralize the charge of the pentacovalent intermediate and/or stabilize the 3' leaving group. Asn212 and His309 also interact with DNA (interactions not shown) and these residues, when substituted have approximately 10^4-fold [40] and 13-fold [23] reductions in DNA-binding affinity, respectively. (b) Model of an Ape1-metal-DNA complex. A stereo image of a modeled complex of Ape1, bound metal ions and abasic DNA is shown. Selected Ape1 side-chains (colored by atom) and the associated metal ions (pink) are from the form III structure and the DNA (blue) is from PDB entry 1DEW [27]. The protein structures were superimposed using the backbone atoms of residues 43-318 using GEM [30] and the Figure was generated using InsightII (BIOSYM/Molecular Simulations, Inc., San Diego, CA). The positions of the scissile bond (P--O3') and the abasic sugar ring (AP) are indicated.
 
  The above figures are reprinted by permission from Elsevier: J Mol Biol (2001, 307, 1023-1034) copyright 2001.  
  Figures were selected by an automated process.  

Literature references that cite this PDB file's key reference

  PubMed id Reference
20808930 A.Gelin, M.Redrejo-Rodríguez, J.Laval, O.S.Fedorova, M.Saparbaev, and A.A.Ishchenko (2010).
Genetic and biochemical characterization of human AP endonuclease 1 mutants deficient in nucleotide incision repair activity.
  PLoS One, 5, 0.  
19888678 B.A.Manvilla, K.M.Varney, and A.C.Drohat (2010).
Chemical shift assignments for human apurinic/apyrimidinic endonuclease 1.
  Biomol NMR Assign, 4, 5-8.  
20084532 C.B.Prasannan, F.Xie, and C.M.Dupureur (2010).
Characterizing metalloendonuclease mixed metal complexes by global kinetic analysis.
  J Biol Inorg Chem, 15, 533-545.  
20706766 G.Tell, D.Fantini, and F.Quadrifoglio (2010).
Understanding different functions of mammalian AP endonuclease (APE1) as a promising tool for cancer treatment.
  Cell Mol Life Sci, 67, 3589-3608.  
19764832 M.Luo, H.He, M.R.Kelley, and M.M.Georgiadis (2010).
Redox regulation of DNA repair: implications for human health and cancer therapeutic development.
  Antioxid Redox Signal, 12, 1247-1269.  
20616014 P.A.Nair, P.Smith, and S.Shuman (2010).
Structure of bacterial LigD 3'-phosphoesterase unveils a DNA repair superfamily.
  Proc Natl Acad Sci U S A, 107, 12822-12827.
PDB codes: 3n9b 3n9d
19968858 W.M.Li, T.Barnes, and C.H.Lee (2010).
Endoribonucleases--enzymes gaining spotlight in mRNA metabolism.
  FEBS J, 277, 627-641.  
18715143 A.Bapat, M.L.Fishel, and M.R.Kelley (2009).
Going ape as an approach to cancer therapeutics.
  Antioxid Redox Signal, 11, 651-668.  
19188445 C.Vascotto, D.Fantini, M.Romanello, L.Cesaratto, M.Deganuto, A.Leonardi, J.P.Radicella, M.R.Kelley, C.D'Ambrosio, A.Scaloni, F.Quadrifoglio, and G.Tell (2009).
APE1/Ref-1 interacts with NPM1 within nucleoli and plays a role in the rRNA quality control process.
  Mol Cell Biol, 29, 1834-1854.  
19123919 S.T.Mundle, J.C.Delaney, J.M.Essigmann, and P.R.Strauss (2009).
Enzymatic mechanism of human apurinic/apyrimidinic endonuclease against a THF AP site model substrate.
  Biochemistry, 48, 19-26.  
19181704 V.M.Castillo-Acosta, L.M.Ruiz-Pérez, W.Yang, D.González-Pacanowska, and A.E.Vidal (2009).
Identification of a residue critical for the excision of 3'-blocking ends in apurinic/apyrimidinic endonucleases of the Xth family.
  Nucleic Acids Res, 37, 1829-1842.  
18436236 A.K.Mantha, N.Oezguen, K.K.Bhakat, T.Izumi, W.Braun, and S.Mitra (2008).
Unusual role of a cysteine residue in substrate binding and activity of human AP-endonuclease 1.
  J Mol Biol, 379, 28-37.  
18576638 A.S.Lipton, R.W.Heck, S.Primak, D.R.McNeill, D.M.Wilson, and P.D.Ellis (2008).
Characterization of Mg2+ binding to the DNA repair protein apurinic/apyrimidic endonuclease 1 via solid-state 25Mg NMR spectroscopy.
  J Am Chem Soc, 130, 9332-9341.  
18579163 M.M.Georgiadis, M.Luo, R.K.Gaur, S.Delaplane, X.Li, and M.R.Kelley (2008).
Evolution of the redox function in mammalian apurinic/apyrimidinic endonuclease.
  Mutat Res, 643, 54-63.
PDB codes: 2o3c 2o3h
18522277 N.S.Dyrkheeva, S.N.Khodyreva, and O.I.Lavrik (2008).
[Quantitative parameters of the 3'-5'-exonuclease reaction of human apurinic/apyrimidinic endonuclease 1 and DNA with single-strand breaks containing dYMP or their modified analogues]
  Bioorg Khim, 34, 210-219.  
18393760 N.S.Dyrkheeva, S.N.Khodyreva, and O.I.Lavrik (2008).
Interaction of APE1 and other repair proteins with DNA duplexes imitating intermediates of DNA repair and replication.
  Biochemistry (Mosc), 73, 261-272.  
17013835 D.R.McNeill, H.K.Wong, A.Narayana, and D.M.Wilson (2007).
Lead promotes abasic site accumulation and co-mutagenesis in mammalian cells by inhibiting the major abasic endonuclease Ape1.
  Mol Carcinog, 46, 91-99.  
17308914 G.A.Papadakos, H.Nastri, P.Riggs, and C.M.Dupureur (2007).
Uncoupling metallonuclease metal ion binding sites via nudge mutagenesis.
  J Biol Inorg Chem, 12, 557-569.  
17699520 K.Rogers, G.Gao, and L.Simpson (2007).
Uridylate-specific 3' 5'-exoribonucleases involved in uridylate-deletion RNA editing in trypanosomatid mitochondria.
  J Biol Chem, 282, 29073-29080.  
17427952 N.Oezguen, C.H.Schein, S.R.Peddi, T.D.Power, T.Izumi, and W.Braun (2007).
A "moving metal mechanism" for substrate cleavage by the DNA repair endonuclease APE-1.
  Proteins, 68, 313-323.  
17724035 R.L.Maher, and L.B.Bloom (2007).
Pre-steady-state kinetic characterization of the AP endonuclease activity of human AP endonuclease 1.
  J Biol Chem, 282, 30577-30585.  
16780580 I.S.Mian, E.A.Worthey, and R.Salavati (2006).
Taking U out, with two nucleases?
  BMC Bioinformatics, 7, 305.  
15155853 A.B.Guliaev, B.Hang, and B.Singer (2004).
Structural insights by molecular dynamics simulations into specificity of the major human AP endonuclease toward the benzene-derived DNA adduct, pBQ-C.
  Nucleic Acids Res, 32, 2844-2852.  
15499577 B.Hang (2004).
Repair of exocyclic DNA adducts: rings of complexity.
  Bioessays, 26, 1195-1208.  
  15159209 D.R.McNeill, A.Narayana, H.K.Wong, and D.M.Wilson (2004).
Inhibition of Ape1 nuclease activity by lead, iron, and cadmium.
  Environ Health Perspect, 112, 799-804.  
14704345 L.Gros, A.A.Ishchenko, H.Ide, R.H.Elder, and M.K.Saparbaev (2004).
The major human AP endonuclease (Ape1) is involved in the nucleotide incision repair pathway.
  Nucleic Acids Res, 32, 73-81.  
15459284 N.G.Beloglazova, O.O.Kirpota, K.V.Starostin, A.A.Ishchenko, V.I.Yamkovoy, D.O.Zharkov, K.T.Douglas, and G.A.Nevinsky (2004).
Thermodynamic, kinetic and structural basis for recognition and repair of abasic sites in DNA by apurinic/apyrimidinic endonuclease from human placenta.
  Nucleic Acids Res, 32, 5134-5146.  
15274918 O.Weichenrieder, K.Repanas, and A.Perrakis (2004).
Crystal structure of the targeting endonuclease of the human LINE-1 retrotransposon.
  Structure, 12, 975-986.
PDB code: 1vyb
14602897 E.A.Worthey, A.Schnaufer, I.S.Mian, K.Stuart, and R.Salavati (2003).
Comparative analysis of editosome proteins in trypanosomatids.
  Nucleic Acids Res, 31, 6392-6408.  
12624104 K.M.Chou, and Y.C.Cheng (2003).
The exonuclease activity of human apurinic/apyrimidinic endonuclease (APE1). Biochemical properties and inhibition by the natural dinucleotide Gp4G.
  J Biol Chem, 278, 18289-18296.  
12694197 M.Miertzschke, and T.Greiner-Stöffele (2003).
The xthA gene product of Archaeoglobus fulgidus is an unspecific DNase.
  Eur J Biochem, 270, 1838-1849.  
12496295 T.Mordasini, A.Curioni, and W.Andreoni (2003).
Why do divalent metal ions either promote or inhibit enzymatic reactions? The case of BamHI restriction endonuclease from combined quantum-classical simulations.
  J Biol Chem, 278, 4381-4384.  
11707423 A.E.Vidal, S.Boiteux, I.D.Hickson, and J.P.Radicella (2001).
XRCC1 coordinates the initial and late stages of DNA abasic site repair through protein-protein interactions.
  EMBO J, 20, 6530-6539.  
The most recent references are shown first. Citation data come partly from CiteXplore and partly from an automated harvesting procedure. Note that this is likely to be only a partial list as not all journals are covered by either method. However, we are continually building up the citation data so more and more references will be included with time. Where a reference describes a PDB structure, the PDB codes are shown on the right.