PDBsum entry 1a31

Isomerase/DNA

PDB id: 1a31

Contents

Protein chain

458 a.a. *

DNA/RNA

Waters ×409

* Residue conservation analysis

PDB id:

1a31

Name:

Isomerase/DNA

Title:

Human reconstituted DNA topoisomerase i in covalent complex with a 22 base pair DNA duplex

Structure:

DNA (5'- d( Ap Ap Ap Ap Ap Gp Ap Cp 5Iup 5Iu Tp Gp Ap Ap Ap Ap Ap 5Iup 5Iup 5I up 5Iup T)-3'). Chain: c. Engineered: yes. DNA (5'- d( Ap Ap Ap Ap Ap Tp 5Iup 5Iup 5Iup 5Iup Cp Ap Ap Ap Gp Tp Cp Tp Tp T p Tp T)-3'). Chain: d.

Source:

Synthetic: yes. Homo sapiens. Human. Organism_taxid: 9606. Cellular_location: nucleus. Expressed in: spodoptera frugiperda. Expression_system_taxid: 7108.

Biol. unit:

Monomer (from PDB file)

Resolution:

2.10Å R-factor: 0.247 R-free: 0.310

Authors:

M.R.Redinbo,L.Stewart,P.Kuhn,J.J.Champoux,W.G.J.Hol

Key ref:

M.R.Redinbo et al. (1998). Crystal structures of human topoisomerase I in covalent and noncovalent complexes with DNA. Science, 279, 1504-1513. PubMed id: 9488644 DOI: 10.1126/science.279.5356.1504

Date:

27-Jan-98 Release date: 28-Aug-98

Protein chain

P11387  (TOP1_HUMAN) -  DNA topoisomerase 1 from Homo sapiens

Seq: 765 a.a.

Struc: 458 a.a.

DNA/RNA chains

A-A-A-A-A-G-A-C-5IU-5IU-T-G-A-A-A-A-A-5IU-5IU-5IU-5IU-T 22 bases

A-A-A-A-A-T-5IU-5IU-5IU-5IU-C-A-A-A-G-T-C-T-T-T-T-T 22 bases

Enzyme reactions

• Enzyme class: E.C.5.6.2.1 - Dna topoisomerase.

DOI no: 10.1126/science.279.5356.1504

Science 279:1504-1513 (1998)

PubMed id: 9488644

Crystal structures of human topoisomerase I in covalent and noncovalent complexes with DNA.

M.R.Redinbo, L.Stewart, P.Kuhn, J.J.Champoux, W.G.Hol.

ABSTRACT

Topoisomerases I promote the relaxation of DNA superhelical tension by introducing a transient single-stranded break in duplex DNA and are vital for the processes of replication, transcription, and recombination. The crystal structures at 2.1 and 2.5 angstrom resolution of reconstituted human topoisomerase I comprising the core and carboxyl-terminal domains in covalent and noncovalent complexes with 22-base pair DNA duplexes reveal an enzyme that "clamps" around essentially B-form DNA. The core domain and the first eight residues of the carboxyl-terminal domain of the enzyme, including the active-site nucleophile tyrosine-723, share significant structural similarity with the bacteriophage family of DNA integrases. A binding mode for the anticancer drug camptothecin is proposed on the basis of chemical and biochemical information combined with these three-dimensional structures of topoisomerase I-DNA complexes.

Selected figure(s)

Figure 2.
Fig. 2. Structural similarities between human topo I and HP1 integrase. (A) The sequence and secondary structural elements of reconstituted human topo I are indicated in the standard coloring scheme of the domain architecture of the enzyme (Fig. 1A), and^ the structurally similar regions of HP1 integrase are shown in red with gray background. Catalytically relevant residues of human topo I are highlighted in cyan, and the positions of known CPT-resistant mutations in human, hamster, and yeast topoisomerases I are shown in gray. -Helices 18 and 19 are not depicted because these correspond^ to the linker domain (20), which is not present in the reconstituted^ enzyme. (B) Stereoview of the superposition of core subdomain III (red) and the COOH-terminal domain (green) of human topo I and bacteriophage HP1 integrase (gray) (38). The active-site^ residues of each enzyme are shown, with the human topo I residues in cyan and the integrase residues in gray. Helices 8, 10, 15, and 17 of core subdomain III of topo I are also indicated. There^ is no structural equivalent in the integrase for the topo I COOH-terminal domain past the first eight residues, which contain the catalytic^ Tyr723. The C positions of the active-site residues Arg488 and Arg590 of topo I (20) superimpose within 0.6 and 1.9 Å, respectively, of the C positions of Arg207 and Arg283 in the integrase. His306 of the integrase superimposes within 3.3 Å on His632 of human topo I, but the putative catalytic His280 of the integrase superimposes on a noncatalytic residue of human topo I, Lys587. Abbreviations for the amino acid residues are as follows: A, Ala; C, Cys; D, Asp; E, Glu; F, Phe; G, Gly; H, His; I, Ile; K, Lys; L, Leu; M, Met; N, Asn; P, Pro; Q, Gln; R, Arg; S, Ser; T, Thr; V, Val; W, Trp; and Y, Tyr.

Figure 6.
Fig. 6. Proposed CPT binding mode. (A) A schematic representation of the key hydrogen bond and ring-stacking interactions made between the human topo I-DNA covalent complex and CPT in the proposed CPT binding mode. The atomic nomenclature for CPT is also indicated. (B) Stereoview of the proposed binding mode of CPT to the covalent human topo I DNA complex. The active^ lactone form of CPT (20-S-camptothecin, in green) is shown stacked^ between the terminal +1 guanine nucleotide from the cleaved strand^ (+1 Gua, in yellow, which is reoriented from the observed position as described below), and the side chain of Asn722, which provides interactions with the A-ring of CPT (the cleaved^ strand is rendered in light and dark magenta upstream and downstream of the cleavage site, respectively). The carbonyl oxygen at the^ 17 position in CPT makes a hydrogen bond with the NH[2] group on the pyrimidine ring of the +1 cytosine. The side chains of active-site^ residues Tyr723, Arg488, and Arg590 are shown in cyan. The side chain residues that, if singly mutated, result in a CPT-resistant phenotype [Phe^361, Gly363, and Arg364 of region 1 (see text); Asp533 and Asn722 of region 2] are shown in tan. The side chain conformations of^ Arg364 and Asp533 have been altered slightly from the final structure of the covalent complex to allow for optimal hydrogen bonding to the double-bonded^ lactone oxygen and the hydroxyl at the 20-S chiral center of CPT, respectively. Modifications to the 10 and 11 positions of CPT may require some minor shifts in the positions of residues Lys720 and Leu721 of topo I, which exhibit relatively high temperature factors (for example, 55 to 65 Å2) in the structure of the covalent complex. The proposed conformation of the +1 Gua nucleotide was inspired by flipped-out bases observed^ experimentally by Sussman and co-workers (62), but was further optimized by rotations about bonds in the intact phosphate between the +1 and +2 nucleotides. Because this base is now a terminal nucleotide in the cleaved strand, it is less contrained by the^ ribose-phosphate backbone and is more free to rotate to positions outside the DNA duplex.

The above figures are reprinted by permission from the AAAs: Science (1998, 279, 1504-1513) copyright 1998.

Figures were selected by an automated process.