PDBsum entry 1krp

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protein dna_rna metals links
Transferase/DNA PDB id
Protein chain
601 a.a. *
_ZN ×4
Waters ×268
* Residue conservation analysis
PDB id:
Name: Transferase/DNA
Title: DNA polymerase i klenow fragment (E.C. mutant/DNA complex
Structure: DNA (5'-d(p Tp Tp Pst)-3'). Chain: b. Engineered: yes. Protein (DNA polymerase i klenow fragment . Chain: a. Synonym: large fragment. Engineered: yes. Mutation: yes
Source: Synthetic: yes. Escherichia coli. Organism_taxid: 562. Expressed in: escherichia coli. Expression_system_taxid: 562.
2.20Å     R-factor:   0.190     R-free:   0.255
Authors: C.A.Brautigam,T.A.Steitz
Key ref:
C.A.Brautigam and T.A.Steitz (1998). Structural principles for the inhibition of the 3'-5' exonuclease activity of Escherichia coli DNA polymerase I by phosphorothioates. J Mol Biol, 277, 363-377. PubMed id: 9514742 DOI: 10.1006/jmbi.1997.1586
19-Aug-97     Release date:   25-Feb-98    
Go to PROCHECK summary

Protein chain
Pfam   ArchSchema ?
P00582  (DPO1_ECOLI) -  DNA polymerase I
928 a.a.
601 a.a.*
Key:    PfamA domain  Secondary structure  CATH domain
* PDB and UniProt seqs differ at 1 residue position (black cross)

 Enzyme reactions 
   Enzyme class: E.C.  - DNA-directed Dna polymerase.
[IntEnz]   [ExPASy]   [KEGG]   [BRENDA]
      Reaction: Deoxynucleoside triphosphate + DNA(n) = diphosphate + DNA(n+1)
Deoxynucleoside triphosphate
+ DNA(n)
= diphosphate
+ DNA(n+1)
Molecule diagrams generated from .mol files obtained from the KEGG ftp site
 Gene Ontology (GO) functional annotation 
  GO annot!
  Biological process     nucleobase-containing compound metabolic process   3 terms 
  Biochemical function     nucleic acid binding     4 terms  


DOI no: 10.1006/jmbi.1997.1586 J Mol Biol 277:363-377 (1998)
PubMed id: 9514742  
Structural principles for the inhibition of the 3'-5' exonuclease activity of Escherichia coli DNA polymerase I by phosphorothioates.
C.A.Brautigam, T.A.Steitz.
A two-metal-ion catalytic mechanism has previously been proposed for several phosphoryl-transfer enzymes. In order to extend the structural basis of this mechanism, crystal structures of three single-stranded DNA substrates bound to the 3'-5' exonucleolytic active site of the large fragment of DNA polymerase I from Escherichia coli have been elucidated. The first is a 2.1 A resolution structure of a Michaelis complex between the large fragment (or Klenow fragment, KF) and a single-stranded DNA substrate, stabilized by low pH and flash-freezing. The positions and identities of the catalytic metal ions, a Zn2+ at site A and a Mg2+ at site B, have been clearly established. The structural and kinetic consequences of sulfur substitutions in the scissile phosphate have been explored. A complex with the Rp isomer of phosphorothioate DNA, refined at 2.2 A resolution, shows Zn2+ bound to both metal sites and a mispositioning of the substrate and attacking nucleophile. The complex with the Sp phosphorothioate at 2. 3 A resolution reveals that metal ions do not bind in the active site, having been displaced by a bulky sulfur atom. Steady-state kinetic experiments show that catalyzed hydrolysis of the Rp isomer was reduced only about 15-fold, while no enzyme activity could be detected with the Sp phosphorothioate, consistent with the structural observations. Furthermore, Mn2+ could not rescue the activity of the exonuclease on the Sp phosphorothioate. Taken together, these studies confirm and extend the proposed two-metal-ion exonuclease mechanism and provide a structural context to explain the effects of sulfur substitutions on this and other phosphoryl-transfer enzymes. These experiments also suggest that the possibility of metal-ion exclusion be taken into account when interpreting the results of Mn2+ rescue experiments.
  Selected figure(s)  
Figure 2.
Figure 2. the configurations of the oxygen (or sulfur) atoms about the scissile phosphate of normal or phos- phorothioate DNA. The phosphates are shown in the orientation that will occur in all other Figures. (a) Nor- mal, or all-oxygen phosphate. The pro-R and pro-S pos- itions are marked. The negative charge is distributed between the non-bridging oxygens. (b) Rp phosphor- othioate phosphate. Note that the sulfur atom has only a single covalent bond to the phosphorus atom and is negatively charged. The pro-S oxygen features a double bond to phosphorus. (c) The Sp phosphorothioate.
Figure 5.
Figure 5. Schematic drawing of the R isomer structure. The same color- ing scheme as in Figure 4 is used, with the pro-R sulfur and ``attack'' water highlighted in yellow and purple, respectively. The two Zn ions are about 4.0 Å apart.
  The above figures are reprinted by permission from Elsevier: J Mol Biol (1998, 277, 363-377) copyright 1998.  
  Figures were selected by an automated process.  

Literature references that cite this PDB file's key reference

  PubMed id Reference
21496642 J.Orans, E.A.McSweeney, R.R.Iyer, M.A.Hast, H.W.Hellinga, P.Modrich, and L.S.Beese (2011).
Structures of human exonuclease 1 DNA complexes suggest a unified mechanism for nuclease family.
  Cell, 145, 212-223.
PDB codes: 3qe9 3qea 3qeb
20854710 W.Yang (2011).
Nucleases: diversity of structure, function and mechanism.
  Q Rev Biophys, 44, 1.  
20706627 G.Pastor-Palacios, E.Azuara-Liceaga, and L.G.Brieba (2010).
A nuclear family A DNA polymerase from Entamoeba histolytica bypasses thymine glycol.
  PLoS Negl Trop Dis, 4, e786.  
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The use of divalent metal ions by type II topoisomerases.
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20589414 M.Wojcik, and W.J.Stec (2010).
The effect of divalent cations on the catalytic activity of the human plasma 3'-exonuclease.
  Biometals, 23, 1113-1121.  
19915588 A.Schwartz, M.Rabhi, F.Jacquinot, E.Margeat, A.R.Rahmouni, and M.Boudvillain (2009).
A stepwise 2'-hydroxyl activation mechanism for the bacterial transcription termination factor Rho helicase.
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19619558 C.A.Wakeman, A.Ramesh, and W.C.Winkler (2009).
Multiple metal-binding cores are required for metalloregulation by M-box riboswitch RNAs.
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19863108 Y.Santoso, and A.N.Kapanidis (2009).
Probing biomolecular structures and dynamics of single molecules using in-gel alternating-laser excitation.
  Anal Chem, 81, 9561-9570.  
18230765 A.Lagunavicius, Z.Kiveryte, V.Zimbaite-Ruskuliene, T.Radzvilavicius, and A.Janulaitis (2008).
Duality of polynucleotide substrates for Phi29 DNA polymerase: 3'-->5' RNase activity of the enzyme.
  RNA, 14, 503-513.  
18769882 B.Nawrot, N.Paul, B.Rebowska, and W.J.Stec (2008).
Significance of stereochemistry of 3'-terminal phosphorothioate-modified primer in DNA polymerase-mediated chain extension.
  Mol Biotechnol, 40, 119-126.  
18653531 J.E.Deweese, A.B.Burgin, and N.Osheroff (2008).
Human topoisomerase IIalpha uses a two-metal-ion mechanism for DNA cleavage.
  Nucleic Acids Res, 36, 4883-4893.  
18975918 J.K.Lassila, and D.Herschlag (2008).
Promiscuous sulfatase activity and thio-effects in a phosphodiesterase of the alkaline phosphatase superfamily.
  Biochemistry, 47, 12853-12859.  
18780819 M.Brucet, J.Querol-Audí, K.Bertlik, J.Lloberas, I.Fita, and A.Celada (2008).
Structural and biochemical studies of TREX1 inhibition by metals. Identification of a new active histidine conserved in DEDDh exonucleases.
  Protein Sci, 17, 2059-2069.
PDB codes: 3b6o 3b6p
18808119 P.A.Sigala, D.A.Kraut, J.M.Caaveiro, B.Pybus, E.A.Ruben, D.Ringe, G.A.Petsko, and D.Herschlag (2008).
Testing geometrical discrimination within an enzyme active site: constrained hydrogen bonding in the ketosteroid isomerase oxyanion hole.
  J Am Chem Soc, 130, 13696-13708.
PDB codes: 2inx 3cpo
18219121 R.D.Busam (2008).
Structure of Escherichia coli exonuclease I in complex with thymidine 5'-monophosphate.
  Acta Crystallogr D Biol Crystallogr, 64, 206-210.
PDB code: 2qxf
17452359 A.T.Jonstrup, K.R.Andersen, L.B.Van, and D.E.Brodersen (2007).
The 1.4-A crystal structure of the S. pombe Pop2p deadenylase subunit unveils the configuration of an active enzyme.
  Nucleic Acids Res, 35, 3153-3164.
PDB code: 2p51
17250742 B.Nawrot, K.Widera, M.Wojcik, B.Rebowska, G.Nowak, and W.J.Stec (2007).
Mapping of the functional phosphate groups in the catalytic core of deoxyribozyme 10-23.
  FEBS J, 274, 1062-1072.  
17517631 C.Ferrer-Orta, A.Arias, R.Pérez-Luque, C.Escarmís, E.Domingo, and N.Verdaguer (2007).
Sequential structures provide insights into the fidelity of RNA replication.
  Proc Natl Acad Sci U S A, 104, 9463-9468.
PDB codes: 2e9r 2e9t 2e9z 2ec0
17640918 G.Luo, M.Wang, W.H.Konigsberg, and X.S.Xie (2007).
Single-molecule and ensemble fluorescence assays for a functionally important conformational change in T7 DNA polymerase.
  Proc Natl Acad Sci U S A, 104, 12610-12615.  
17267608 G.Sasnauskas, B.A.Connolly, S.E.Halford, and V.Siksnys (2007).
Site-specific DNA transesterification catalyzed by a restriction enzyme.
  Proc Natl Acad Sci U S A, 104, 2115-2120.  
18059450 I.Artsimovitch, and D.G.Vassylyev (2007).
Merging the RNA and DNA worlds.
  Nat Struct Mol Biol, 14, 1122-1123.  
17229737 J.M.Choi, S.Y.Kang, W.J.Bae, K.S.Jin, M.Ree, and Y.Cho (2007).
Probing the roles of active site residues in the 3'-5' exonuclease of the Werner syndrome protein.
  J Biol Chem, 282, 9941-9951.  
16552775 A.P.So, R.F.Turner, and C.A.Haynes (2006).
Minimizing loss of sequence information in SAGE ditags by modulating the temperature dependent 3' --> 5' exonuclease activity of DNA polymerases on 3'-terminal isoheptyl amino groups.
  Biotechnol Bioeng, 94, 54-65.  
16912289 D.Lim, G.G.Gregorio, C.Bingman, E.Martinez-Hackert, W.A.Hendrickson, and S.P.Goff (2006).
Crystal structure of the moloney murine leukemia virus RNase H domain.
  J Virol, 80, 8379-8389.
PDB code: 2hb5
16699515 J.A.Jansen, T.J.McCarthy, G.A.Soukup, and J.K.Soukup (2006).
Backbone and nucleobase contacts to glucosamine-6-phosphate in the glmS ribozyme.
  Nat Struct Mol Biol, 13, 517-523.  
16622405 J.J.Perry, S.M.Yannone, L.G.Holden, C.Hitomi, A.Asaithamby, S.Han, P.K.Cooper, D.J.Chen, and J.A.Tainer (2006).
WRN exonuclease structure and molecular mechanism imply an editing role in DNA end processing.
  Nat Struct Mol Biol, 13, 414-422.
PDB codes: 2fbt 2fbv 2fbx 2fby 2fc0
16882719 S.F.Midtgaard, J.Assenholt, A.T.Jonstrup, L.B.Van, T.H.Jensen, and D.E.Brodersen (2006).
Structure of the nuclear exosome component Rrp6p reveals an interplay between the active site and the HRDC domain.
  Proc Natl Acad Sci U S A, 103, 11898-11903.
PDB codes: 2hbj 2hbk 2hbl 2hbm
16600865 W.Yang, J.Y.Lee, and M.Nowotny (2006).
Making and breaking nucleic acids: two-Mg2+-ion catalysis and substrate specificity.
  Mol Cell, 22, 5.  
16027443 P.Haeberli, I.Berger, P.S.Pallan, and M.Egli (2005).
Syntheses of 4'-thioribonucleosides and thermodynamic stability and crystal structure of RNA oligomers with incorporated 4'-thiocytosine.
  Nucleic Acids Res, 33, 3965-3975.
PDB code: 2a0p
15107495 A.Lupták, and J.A.Doudna (2004).
Distinct sites of phosphorothioate substitution interfere with folding and splicing of the Anabaena group I intron.
  Nucleic Acids Res, 32, 2272-2280.  
15375162 I.Benzaghou, I.Bougie, and M.Bisaillon (2004).
Effect of metal ion binding on the structural stability of the hepatitis C virus RNA polymerase.
  J Biol Chem, 279, 49755-49761.  
15102449 M.Steiniger-White, I.Rayment, and W.S.Reznikoff (2004).
Structure/function insights into Tn5 transposition.
  Curr Opin Struct Biol, 14, 50-57.
PDB code: 1mus
15130133 P.A.Pribil, S.J.Wardle, and D.B.Haniford (2004).
Enhancement and rescue of target capture in Tn10 transposition by site-specific modifications in target DNA.
  Mol Microbiol, 52, 1173-1186.  
15358788 Y.G.Ren, L.A.Kirsebom, and A.Virtanen (2004).
Coordination of divalent metal ions in the active site of poly(A)-specific ribonuclease.
  J Biol Chem, 279, 48702-48706.  
15576350 Y.S.Kovacheva, S.B.Tzokov, I.A.Murray, and J.A.Grasby (2004).
The role of phosphate groups in the VS ribozyme-substrate interaction.
  Nucleic Acids Res, 32, 6240-6250.  
14704353 Y.Shen, X.F.Tang, H.Yokoyama, E.Matsui, and I.Matsui (2004).
A 21-amino acid peptide from the cysteine cluster II of the family D DNA polymerase from Pyrococcus horikoshii stimulates its nuclease activity which is Mre11-like and prefers manganese ion as the cofactor.
  Nucleic Acids Res, 32, 158-168.  
12851928 C.P.Da Costa, A.Okruszek, and H.Sigel (2003).
Complex formation of divalent metal ions with uridine 5'-O-thiomonophosphate or methyl thiophosphate: comparison of complex stabilities with those of the parent phosphate ligands.
  Chembiochem, 4, 593-602.  
12560510 D.Di Giusto, and G.C.King (2003).
Single base extension (SBE) with proofreading polymerases and phosphorothioate primers: improved fidelity in single-substrate assays.
  Nucleic Acids Res, 31, e7.  
12458224 I.Bougie, S.Charpentier, and M.Bisaillon (2003).
Characterization of the metal ion binding properties of the hepatitis C virus RNA polymerase.
  J Biol Chem, 278, 3868-3875.  
12727889 V.Sosunov, E.Sosunova, A.Mustaev, I.Bass, V.Nikiforov, and A.Goldfarb (2003).
Unified two-metal mechanism of RNA synthesis and degradation by RNA polymerase.
  EMBO J, 22, 2234-2244.  
11980722 E.L.Christian, N.M.Kaye, and M.E.Harris (2002).
Evidence for a polynuclear metal ion binding site in the catalytic domain of ribonuclease P RNA.
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11896402 S.Lovell, I.Y.Goryshin, W.R.Reznikoff, and I.Rayment (2002).
Two-metal active site binding of a Tn5 transposase synaptic complex.
  Nat Struct Biol, 9, 278-281.
PDB codes: 1l1a 1mur 4dm0
12166648 S.M.Crary, J.C.Kurz, and C.A.Fierke (2002).
Specific phosphorothioate substitutions probe the active site of Bacillus subtilis ribonuclease P.
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12087102 V.M.Petrov, S.S.Ng, and J.D.Karam (2002).
Protein determinants of RNA binding by DNA polymerase of the T4-related bacteriophage RB69.
  J Biol Chem, 277, 33041-33048.  
11900537 W.C.Lam, E.H.Thompson, O.Potapova, X.C.Sun, C.M.Joyce, and D.P.Millar (2002).
3'-5' exonuclease of Klenow fragment: role of amino acid residues within the single-stranded DNA binding region in exonucleolysis and duplex DNA melting.
  Biochemistry, 41, 3943-3951.  
11742007 Y.G.Ren, J.Martínez, and A.Virtanen (2002).
Identification of the active site of poly(A)-specific ribonuclease by site-directed mutagenesis and Fe(2+)-mediated cleavage.
  J Biol Chem, 277, 5982-5987.  
11861910 Z.Morávek, S.Neidle, and B.Schneider (2002).
Protein and drug interactions in the minor groove of DNA.
  Nucleic Acids Res, 30, 1182-1191.  
11180387 P.Guga, K.Domański, and W.J.Stec (2001).
Oxathiaphospholane Approach to the Synthesis of P-Chiral, Isotopomeric Deoxy(ribonucleoside phosphorothioate)s and Phosphates Labeled with an Oxygen Isotope This work was financially supported by the State Committee for Scientific Research (KBN, Poland, Grant 4P05F00617, to W.J.S.), and, in part, by the Human Science Promotion Foundation (Japan, to H. Takaku and W.J.S.).
  Angew Chem Int Ed Engl, 40, 610-613.  
11170438 S.Cogoi, V.Rapozzi, F.Quadrifoglio, and L.Xodo (2001).
Anti-gene effect in live cells of AG motif triplex-forming oligonucleotides containing an increasing number of phosphorothioate linkages.
  Biochemistry, 40, 1135-1143.  
11328865 Y.Takagi, M.Warashina, W.J.Stec, K.Yoshinari, and K.Taira (2001).
Recent advances in the elucidation of the mechanisms of action of ribozymes.
  Nucleic Acids Res, 29, 1815-1834.  
10847684 A.K.Kennedy, D.B.Haniford, and K.Mizuuchi (2000).
Single active site catalysis of the successive phosphoryl transfer steps by DNA transposases: insights from phosphorothioate stereoselectivity.
  Cell, 101, 295-305.  
10786845 B.C.Thomas, X.Li, and P.Gegenheimer (2000).
Chloroplast ribonuclease P does not utilize the ribozyme-type pre-tRNA cleavage mechanism.
  RNA, 6, 545-553.  
10625479 D.Dertinger, L.S.Behlen, and O.C.Uhlenbeck (2000).
Using phosphorothioate-substituted RNA to investigate the thermodynamic role of phosphates in a sequence specific RNA-protein complex.
  Biochemistry, 39, 55-63.  
10786842 E.L.Christian, N.M.Kaye, and M.E.Harris (2000).
Helix P4 is a divalent metal ion binding site in the conserved core of the ribonuclease P ribozyme.
  RNA, 6, 511-519.  
  10805163 F.Eckstein (2000).
Phosphorothioate oligodeoxynucleotides: what is their origin and what is unique about them?
  Antisense Nucleic Acid Drug Dev, 10, 117-121.  
10637323 J.M.Warnecke, E.J.Sontheimer, J.A.Piccirilli, and R.K.Hartmann (2000).
Active site constraints in the hydrolysis reaction catalyzed by bacterial RNase P: analysis of precursor tRNAs with a single 3'-S-phosphorothiolate internucleotide linkage.
  Nucleic Acids Res, 28, 720-727.  
10692410 K.Klumpp, L.Doan, N.A.Roberts, and B.Handa (2000).
RNA and DNA hydrolysis are catalyzed by the influenza virus endonuclease.
  J Biol Chem, 275, 6181-6188.  
10734192 K.Yoshinari, and K.Taira (2000).
A further investigation and reappraisal of the thio effect in the cleavage reaction catalyzed by a hammerhead ribozyme.
  Nucleic Acids Res, 28, 1730-1742.  
11112542 M.E.Glasner, C.C.Yen, E.H.Ekland, and D.P.Bartel (2000).
Recognition of nucleoside triphosphates during RNA-catalyzed primer extension.
  Biochemistry, 39, 15556-15562.  
10864040 S.O.Shan, and D.Herschlag (2000).
An unconventional origin of metal-ion rescue and inhibition in the Tetrahymena group I ribozyme reaction.
  RNA, 6, 795-813.  
10103058 C.M.Dupureur, and L.M.Hallman (1999).
Effects of divalent metal ions on the activity and conformation of native and 3-fluorotyrosine-PvuII endonucleases.
  Eur J Biochem, 261, 261-268.  
10047577 J.Jäger, and J.D.Pata (1999).
Getting a grip: polymerases and their substrate complexes.
  Curr Opin Struct Biol, 9, 21-28.  
10588690 M.Teplova, S.T.Wallace, V.Tereshko, G.Minasov, A.M.Symons, P.D.Cook, M.Manoharan, and M.Egli (1999).
Structural origins of the exonuclease resistance of a zwitterionic RNA.
  Proc Natl Acad Sci U S A, 96, 14240-14245.
PDB codes: 1d8y 1d9d 1d9f 1d9h
10580468 S.Basu, and S.A.Strobel (1999).
Thiophilic metal ion rescue of phosphorothioate interference within the Tetrahymena ribozyme P4-P6 domain.
  RNA, 5, 1399-1407.  
10572011 S.Wang, K.Karbstein, A.Peracchi, L.Beigelman, and D.Herschlag (1999).
Identification of the hammerhead ribozyme metal ion binding site responsible for rescue of the deleterious effect of a cleavage site phosphorothioate.
  Biochemistry, 38, 14363-14378.  
10364165 T.A.Steitz (1999).
DNA polymerases: structural diversity and common mechanisms.
  J Biol Chem, 274, 17395-17398.  
10545321 Y.Zhao, D.Jeruzalmi, I.Moarefi, L.Leighton, R.Lasken, and J.Kuriyan (1999).
Crystal structure of an archaebacterial DNA polymerase.
  Structure, 7, 1189-1199.
PDB codes: 1d5a 1qqc
9814764 C.S.Vörtler, O.Fedorova, T.Persson, U.Kutzke, and F.Eckstein (1998).
Determination of 2'-hydroxyl and phosphate groups important for aminoacylation of Escherichia coli tRNAAsp: a nucleotide analogue interference study.
  RNA, 4, 1444-1454.  
9783752 H.Viadiu, and A.K.Aggarwal (1998).
The role of metals in catalysis by the restriction endonuclease BamHI.
  Nat Struct Biol, 5, 910-916.
PDB codes: 2bam 3bam
9843513 M.Boudvillain, and A.M.Pyle (1998).
Defining functional groups, core structural features and inter-domain tertiary contacts essential for group II intron self-splicing: a NAIM analysis.
  EMBO J, 17, 7091-7104.  
9811827 N.C.Horton, K.J.Newberry, and J.J.Perona (1998).
Metal ion-mediated substrate-assisted catalysis in type II restriction endonucleases.
  Proc Natl Acad Sci U S A, 95, 13489-13494.
PDB code: 1bss
9914251 S.Doublié, and T.Ellenberger (1998).
The mechanism of action of T7 DNA polymerase.
  Curr Opin Struct Biol, 8, 704-712.  
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.