PDBsum entry 1r49

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protein dna_rna links
Isomerase/DNA PDB id
Protein chain
548 a.a. *
Waters ×29
* Residue conservation analysis
PDB id:
Name: Isomerase/DNA
Title: Human topoisomerase i (topo70) double mutant k532r/y723f
Structure: 5'- d( Ap Ap Ap Ap Ap Gp Ap Cp Tp Tp Ap Gp Ap Ap Ap Ap Ap Tp Tp Tp Tp T)-3'. Chain: b. Engineered: yes. 5'- d(p Ap Ap Ap Ap Ap Tp Tp Tp Tp Tp Cp Tp Ap Ap Gp Tp Cp Tp T p Tp Tp T)-3'. Chain: c.
Source: Synthetic: yes. Homo sapiens. Human. Organism_taxid: 9606. Gene: top1. Expressed in: spodoptera frugiperda. Expression_system_taxid: 7108.
Biol. unit: Trimer (from PQS)
3.13Å     R-factor:   0.280     R-free:   0.339
Authors: H.Interthal,P.M.Quigley,W.G.Hol,J.J.Champoux
Key ref:
H.Interthal et al. (2004). The role of lysine 532 in the catalytic mechanism of human topoisomerase I. J Biol Chem, 279, 2984-2992. PubMed id: 14594810 DOI: 10.1074/jbc.M309959200
03-Oct-03     Release date:   16-Dec-03    
Go to PROCHECK summary

Protein chain
Pfam   ArchSchema ?
P11387  (TOP1_HUMAN) -  DNA topoisomerase 1
765 a.a.
548 a.a.*
Key:    PfamA domain  Secondary structure  CATH domain
* PDB and UniProt seqs differ at 2 residue positions (black crosses)

 Enzyme reactions 
   Enzyme class: E.C.  - Dna topoisomerase.
[IntEnz]   [ExPASy]   [KEGG]   [BRENDA]
      Reaction: ATP-independent breakage of single-stranded DNA, followed by passage and rejoining.
 Gene Ontology (GO) functional annotation 
  GO annot!
  Cellular component     chromosome   1 term 
  Biological process     DNA topological change   1 term 
  Biochemical function     DNA binding     3 terms  


DOI no: 10.1074/jbc.M309959200 J Biol Chem 279:2984-2992 (2004)
PubMed id: 14594810  
The role of lysine 532 in the catalytic mechanism of human topoisomerase I.
H.Interthal, P.M.Quigley, W.G.Hol, J.J.Champoux.
Based on co-crystal structures of human topoisomerase I with bound DNA, Lys(532) makes a minor groove contact with the strongly preferred thymidine residue at the site of covalent attachment (-1 position). Replacement of Lys(532) with either arginine or alanine has essentially no effect on the sequence preference of the enzyme, indicating that this interaction is not required for the preference for a T at the -1 position. Although both the cleavage and religation activities of the K532R mutant enzyme are reduced, cleavage is reduced to a greater extent than religation. The reverse is true for the K532A mutant enzyme with religation so impaired that the nicked intermediate accumulates during plasmid relaxation assays. Consistent with the shift in the cleavage religation equilibrium toward cleavage for the K532A mutant enzyme, expression of the mutant enzyme in Saccharomyces cerevisiae is cytotoxic, and thus this mutant enzyme mimics the effects of the anticancer drug camptothecin. Cleavage assays with the mutant enzymes using an oligonucleotide containing a 5'-bridging phosphorothiolate indicate that Lys(532) functions as a general acid during cleavage to protonate the leaving 5'-oxygen. It is possible that the contact with the -1 base is important during catalysis to provide positional rigidity to the active site. The corresponding residues in the vaccinia virus topoisomerase and the tyrosine recombinases may have similar critical roles in catalysis.
  Selected figure(s)  
Figure 7.
FIG. 7. Expression of human topoisomerase I K532A in S. cerevisiae. 5 µl of 10-fold serial dilutions (indicated by triangles) of top1 mutant yeast cells containing either empty vector or plasmids encoding WT human topoisomerase I or the K532A mutant were spotted on plates in duplicate. Expression of the proteins is repressed in glucose-containing medium and induced in the presence of galactose.
Figure 9.
FIG. 9. Stereo diagram of the superposition of active sites from topo70 K532R/Y723F and topo70 Y723F. The topo70 K532R/Y723F protein is colored red, and active site residues are shown in ball and stick model with the color scheme of gray for carbon, red for oxygen, and blue for nitrogen. The DNA in complex with topo70 K532R/Y723F is colored gold with the phosphorus atom of the scissile phosphate shown in magenta. The topo70 Y723F structure is colored green for all atoms including the DNA. Distances in angstroms are shown with dashed lines.
  The above figures are reprinted by permission from the ASBMB: J Biol Chem (2004, 279, 2984-2992) copyright 2004.  
  Figures were selected by the author.  

Literature references that cite this PDB file's key reference

  PubMed id Reference
20532182 G.Mancini, I.D'Annessa, A.Coletta, N.Sanna, G.Chillemi, and A.Desideri (2010).
Structural and dynamical effects induced by the anticancer drug topotecan on the human topoisomerase I - DNA complex.
  PLoS One, 5, e10934.  
19472416 C.Punchihewa, M.Carver, and D.Yang (2009).
DNA sequence selectivity of human topoisomerase I-mediated DNA cleavage induced by camptothecin.
  Protein Sci, 18, 1326-1331.  
19767617 P.Fiorani, C.Tesauro, G.Mancini, G.Chillemi, I.D'Annessa, G.Graziani, L.Tentori, A.Muzi, and A.Desideri (2009).
Evidence of the crucial role of the linker domain on the catalytic activity of human topoisomerase I by experimental and simulative characterization of the Lys681Ala mutant.
  Nucleic Acids Res, 37, 6849-6858.  
19740104 Z.Yang, J.F.Carey, and J.J.Champoux (2009).
Mutational analysis of the preferential binding of human topoisomerase I to supercoiled DNA.
  FEBS J, 276, 5906-5919.  
18755053 A.J.Schoeffler, and J.M.Berger (2008).
DNA topoisomerases: harnessing and constraining energy to govern chromosome topology.
  Q Rev Biophys, 41, 41.  
18367446 L.Yakovleva, S.Chen, S.M.Hecht, and S.Shuman (2008).
Chemical and traditional mutagenesis of vaccinia DNA topoisomerase provides insights to cleavage site recognition and transesterification chemistry.
  J Biol Chem, 283, 16093-16103.  
17889664 H.Aihara, W.M.Huang, and T.Ellenberger (2007).
An interlocked dimer of the protelomerase TelK distorts DNA structure for the formation of hairpin telomeres.
  Mol Cell, 27, 901-913.
PDB code: 2v6e
18254239 I.Choi, C.Kim, and S.Choi (2007).
Binding mode analysis of topoisomerase inhibitors, 6-arylamino-7-chloro-quinazoline-5,8-diones, within the cleavable complex of human topoisomerase I and DNA.
  Arch Pharm Res, 30, 1526-1535.  
16505102 C.Marchand, S.Antony, K.W.Kohn, M.Cushman, A.Ioanoviciu, B.L.Staker, A.B.Burgin, L.Stewart, and Y.Pommier (2006).
A novel norindenoisoquinoline structure reveals a common interfacial inhibitor paradigm for ternary trapping of the topoisomerase I-DNA covalent complex.
  Mol Cancer Ther, 5, 287-295.  
16859497 L.Rajeev, A.A.Salyers, and J.F.Gardner (2006).
Characterization of the integrase of NBU1, a Bacteroides mobilizable transposon.
  Mol Microbiol, 61, 978-990.  
15830206 J.B.Leppard, and J.J.Champoux (2005).
Human DNA topoisomerase I: relaxation, roles, and damage control.
  Chromosoma, 114, 75-85.  
15347588 G.Chillemi, M.Redinbo, A.Bruselles, and A.Desideri (2004).
Role of the linker domain and the 203-214 N-terminal residues in the human topoisomerase I DNA complex dynamics.
  Biophys J, 87, 4087-4097.  
15494452 J.J.Vermeersch, S.Christmann-Franck, L.V.Karabashyan, S.Fermandjian, G.Mirambeau, and P.A.Der Garabedian (2004).
Pyridoxal 5'-phosphate inactivates DNA topoisomerase IB by modifying the lysine general acid.
  Nucleic Acids Res, 32, 5649-5657.  
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 code is shown on the right.