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PDBsum entry 2jef
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protein dna_rna ligands metals links
Transferase/DNA PDB id
2jef

 

 

 

 

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JSmol PyMol  
Contents
Protein chain
343 a.a. *
DNA/RNA
Ligands
DGT
Metals
_CA ×3
Waters ×182
* Residue conservation analysis
PDB id:
2jef
Name: Transferase/DNA
Title: The molecular basis of selectivity of nucleotide triphosphate incorporation opposite o6-benzylguanine by sulfolobus solfataricus DNA polymerase iv: steady-state and pre-steady-state and x-ray crystallography of correct and incorrect pairing
Structure: DNA polymerase iv. Chain: a. Synonym: pol iv. Engineered: yes. Other_details: his-tag added to n-terminus. 5'-d( Gp Gp Gp Gp Gp Ap Ap Gp Gp Ap Tp Tp Cp Doc)-3'. Chain: p. Engineered: yes. Other_details: doc indicates a dideoxy-cytosine residue.
Source: Sulfolobus solfataricus. Organism_taxid: 273057. Strain: p2. Expressed in: escherichia coli. Expression_system_taxid: 469008. Synthetic: yes. Synthetic: yes
Resolution:
2.17Å     R-factor:   0.230     R-free:   0.267
Authors: R.L.Eoff,K.C.Angel,I.D.Kosekov,M.Egli,F.P.Guengerich
Key ref:
R.L.Eoff et al. (2007). Molecular basis of selectivity of nucleoside triphosphate incorporation opposite O6-benzylguanine by sulfolobus solfataricus DNA polymerase Dpo4: steady-state and pre-steady-state kinetics and x-ray crystallography of correct and incorrect pairing. J Biol Chem, 282, 13573-13584. PubMed id: 17337730 DOI: 10.1074/jbc.M700656200
Date:
17-Jan-07     Release date:   13-Mar-07    
PROCHECK
Go to PROCHECK summary
 Headers
 References

Protein chain
Pfam   ArchSchema ?
Q97W02  (DPO4_SULSO) -  DNA polymerase IV from Saccharolobus solfataricus (strain ATCC 35092 / DSM 1617 / JCM 11322 / P2)
Seq:
Struc: 		352 a.a.
343 a.a.
Key:    PfamA domain  Secondary structure  CATH domain

DNA/RNA chains
  G-G-G-G-G-A-A-G-G-A-T-T-C-DOC 14 bases
  A-C-BZG-G-A-A-T-C-C-T-T-C-C-C-C-C 16 bases

 Enzyme reactions 
   Enzyme class: E.C.2.7.7.7  - DNA-directed Dna polymerase.
[IntEnz]   [ExPASy]   [KEGG]   [BRENDA]
      Reaction: DNA(n) + a 2'-deoxyribonucleoside 5'-triphosphate = DNA(n+1) + diphosphate
DNA(n)
 + 2'-deoxyribonucleoside 5'-triphosphate
 = DNA(n+1)
 + diphosphate
Molecule diagrams generated from .mol files obtained from the KEGG ftp site

 

 
    Added reference    
 
 
DOI no: 10.1074/jbc.M700656200 J Biol Chem 282:13573-13584 (2007)
PubMed id: 17337730  
 
 
Molecular basis of selectivity of nucleoside triphosphate incorporation opposite O6-benzylguanine by sulfolobus solfataricus DNA polymerase Dpo4: steady-state and pre-steady-state kinetics and x-ray crystallography of correct and incorrect pairing.
R.L.Eoff, K.C.Angel, M.Egli, F.P.Guengerich.
 
  ABSTRACT  
 
Previous work has shown that Sulfolobus solfataricus DNA polymerase Dpo4-catalyzed bypass of O(6)-methylguanine (O(6)-MeG) proceeds largely in an accurate but inefficient manner with a "wobble" base pairing between C and O(6)-MeG (Eoff, R. L., Irimia, A., Egli, M., and Guengerich, F. P. (2007) J. Biol. Chem. 282, 1456-1467). We considered here the bulky lesion O(6)-benzylguanine (O(6)-BzG) in DNA and catalysis by Dpo4. Mass spectrometry analysis of polymerization products revealed that the enzyme bypasses and extends across from O(6)-BzG, with C the major product ( approximately 70%) and some T and A ( approximately 15% each) incorporated opposite the lesion. Steady-state kinetic parameters indicated that Dpo4 was 7-, 5-, and 27-fold more efficient at C incorporation opposite O(6)-BzG than T, A, or G, respectively. In transient state kinetic analysis, the catalytic efficiency was decreased 62-fold for C incorporation opposite O(6)-BzG relative to unmodified DNA. Crystal structures reveal wobble pairing between C and O(6)-BzG. Pseudo-"Watson-Crick" pairing was observed between T and O(6)-BzG. Two other structures illustrate a possible mechanism for the accommodation of a +1 frameshift in the Dpo4 active site. The overall effect of O(6)-BzG is to decrease the efficiency of bypass by roughly an order of magnitude in every case except correct bypass, where the effect is not as pronounced. By comparison, Dpo4 is more accurate but no more efficient than model replicative polymerases, such as bacteriophage T7(-) DNA polymerase and human immunodeficiency virus-1 reverse transcriptase in the polymerization past O(6)-MeG and O(6)-BzG.
 
  Selected figure(s)  
 
Figure 7.
FIGURE 7. Comparison of primer ddC orientations in the O^6-BzG:ddC-1 and O^6-BzG:ddC-2 structures and a view of the O^6-BzG:G active site. A, stereo view of the superimposed (r.m.s. deviation = 0.344) O^6-BzG:ddC-1 (Dpo4 (light blue) and nucleotides and ions (dark blue)) and O^6-BzG:ddC-2 (Dpo4, ions, and nucleotides (orange)) structures. Dpo4 is shown in schematic diagram form for both structures. The incoming dGTP (magenta) from the O^6-BzG:ddC-1 structure is shown in ball and stick form. The 14th nucleotide (dark blue, ddC) from the O^6-BzG:ddC-1 structure forms a wobble pair with O^6-BzG, but the 14th nucleotide (orange, ddC) from the O^6-BzG:ddC-2 ternary complex is moved into a noninstructional conformation. B, stereo view of the O^6-BzG:G active site. Dpo4 (light blue) is shown in schematic diagram form. The last two primer residues, p14C and p15G, and the O^6-BzG lesion are shown in ball and stick form (dark blue). The 14th base in the primer, p14C, is placed in a conformation similar to that observed in the O^6-BzG:ddC-2 structure. The calcium ions (light brown) and incoming dGTP (magenta) are also shown. 		Figure 8.
FIGURE 8. View of the O^6-BzG:T active site superimposed on the wobble O^6-BzG:ddC-1 pair. A, stereo view of the active site from the O^6-BzG:T ternary complex is shown superimposed on the O^6-BzG:ddC-1 structure (r.m.s. deviation = 0.390) with Dpo4 in schematic diagram form (O^6-BzG:T (ruby), O^6-BzG: ddC-1 (light blue)). The O^6-BzG:T pair is shown with cyan carbon atoms, and the O^6-BzG:ddC-1 wobble pair is shown with dark blue carbon atoms. The O^6-BzG:T calcium ions are red, and the O^6-BzG:ddC-1 calcium ions are dark blue. The incoming dGTP from the O^6-BzG:T structure is shown in ball and stick form (magenta). B, the p14T residue (cyan carbon atoms) is shifted up in the active site relative to p14ddC-1 (dark blue carbon atoms), and the benzyl group in the O^6-BzG:T structure is shifted slightly toward the proximal orientation.
 
  The above figures are reprinted by permission from the ASBMB: J Biol Chem (2007, 282, 13573-13584) copyright 2007.  
  Figures were selected by an automated process.  

Literature references that cite this PDB file's key reference

  PubMed id Reference
20123134 J.D.Pata (2010).
Structural diversity of the Y-family DNA polymerases.
  Biochim Biophys Acta, 1804, 1124-1135.  
  20936176 P.Raychaudhury, and A.K.Basu (2010).
Replication Past the γ-Radiation-Induced Guanine-Thymine Cross-Link G[8,5-Me]T by Human and Yeast DNA Polymerase η.
  J Nucleic Acids, 2010, 0.  
  20936119 R.L.Eoff, J.Y.Choi, and F.P.Guengerich (2010).
Mechanistic Studies with DNA Polymerases Reveal Complex Outcomes following Bypass of DNA Damage.
  J Nucleic Acids, 2010, 0.  
19757831 H.A.Dahlmann, V.G.Vaidyanathan, and S.J.Sturla (2009).
Investigating the biochemical impact of DNA damage with structure-based probes: abasic sites, photodimers, alkylation adducts, and oxidative lesions.
  Biochemistry, 48, 9347-9359.  
19059910 H.Zhang, R.L.Eoff, I.D.Kozekov, C.J.Rizzo, M.Egli, and F.P.Guengerich (2009).
Versatility of Y-family Sulfolobus solfataricus DNA polymerase Dpo4 in translesion synthesis past bulky N2-alkylguanine adducts.
  J Biol Chem, 284, 3563-3576.
PDB codes: 2v4s 2v4t 2w8k 2w8l
19542237 H.Zhang, R.L.Eoff, I.D.Kozekov, C.J.Rizzo, M.Egli, and F.P.Guengerich (2009).
Structure-function relationships in miscoding by Sulfolobus solfataricus DNA polymerase Dpo4: guanine N2,N2-dimethyl substitution produces inactive and miscoding polymerase complexes.
  J Biol Chem, 284, 17687-17699.
PDB codes: 2w9a 2w9b 2w9c
19446528 O.Rechkoblit, L.Malinina, Y.Cheng, N.E.Geacintov, S.Broyde, and D.J.Patel (2009).
Impact of conformational heterogeneity of OxoG lesions and their pairing partners on bypass fidelity by Y family polymerases.
  Structure, 17, 725-736.
PDB codes: 3gii 3gij 3gik 3gil 3gim
19397282 P.P.Christov, K.C.Angel, F.P.Guengerich, and C.J.Rizzo (2009).
Replication past the N5-methyl-formamidopyrimidine lesion of deoxyguanosine by DNA polymerases and an improved procedure for sequence analysis of in vitro bypass products by mass spectrometry.
  Chem Res Toxicol, 22, 1086-1095.  
19492857 R.L.Eoff, J.B.Stafford, J.Szekely, C.J.Rizzo, M.Egli, F.P.Guengerich, and L.J.Marnett (2009).
Structural and functional analysis of Sulfolobus solfataricus Y-family DNA polymerase Dpo4-catalyzed bypass of the malondialdehyde-deoxyguanosine adduct.
  Biochemistry, 48, 7079-7088.
PDB codes: 2v4q 2v4r
19124465 S.M.Sherrer, J.A.Brown, L.R.Pack, V.P.Jasti, J.D.Fowler, A.K.Basu, and Z.Suo (2009).
Mechanistic studies of the bypass of a bulky single-base lesion catalyzed by a Y-family DNA polymerase.
  J Biol Chem, 284, 6379-6388.  
18072751 J.C.Delaney, and J.M.Essigmann (2008).
Biological properties of single chemical-DNA adducts: a twenty year perspective.
  Chem Res Toxicol, 21, 232-252.  
18984592 J.W.Beckman, Q.Wang, and F.P.Guengerich (2008).
Kinetic analysis of correct nucleotide insertion by a Y-family DNA polymerase reveals conformational changes both prior to and following phosphodiester bond formation as detected by tryptophan fluorescence.
  J Biol Chem, 283, 36711-36723.  
18988239 N.Böge, M.I.Jacobsen, Z.Szombati, S.Baerns, F.Di Pasquale, A.Marx, and C.Meier (2008).
Synthesis of DNA strands site-specifically damaged by c8-arylamine purine adducts and effects on various DNA polymerases.
  Chemistry, 14, 11194-11208.  
17971991 D.Vineyard, X.Zhang, A.Donnelly, I.Lee, and A.J.Berdis (2007).
Optimization of non-natural nucleotides for selective incorporation opposite damaged DNA.
  Org Biomol Chem, 5, 3623-3630.  
17898175 W.Yang, and R.Woodgate (2007).
What a difference a decade makes: insights into translesion DNA synthesis.
  Proc Natl Acad Sci U S A, 104, 15591-15598.  
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.

 

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