Research article summary (published 27 Sep 2009):

Metal ion interactions in the DNA cleavage/ligation active site of human topoisomerase IIalpha.

Full Abstract

Human topoisomerase IIalpha utilizes a two-metal-ion mechanism for DNA cleavage. One of the metal ions (M(1)(2+)) is believed to make a critical interaction with the 3'-bridging atom of the scissile phosphate, while the other (M(2)(2+)) is believed to interact with a nonbridging oxygen of the scissile phosphate. Based on structural and mutagenesis studies of prokaryotic nucleic acid enzymes, it has been proposed that the active site divalent metal ions interact with type II topoisomerases through a series of conserved acidic amino acid residues. The homologous residues in human topoisomerase IIalpha are E461, D541, D543, and D545. To address the validity of these assignments and to delineate interactions between individual amino acids and M(1)(2+) and M(2)(2+), we individually mutated each of these acidic amino acid residues in topoisomerase IIalpha to either cysteine or alanine. Mutant enzymes displayed a marked loss of catalytic and DNA cleavage activity as well as a reduced affinity for divalent metal ions. Additional experiments determined the ability of wild-type and mutant topoisomerase IIalpha enzymes to cleave an oligonucleotide substrate that contained a sulfur atom in place of the 3'-bridging oxygen of the scissile phosphate in the presence of Mg2+, Mn2+, or Ca2+. On the basis of the results of these studies, we conclude that the four acidic amino acid residues interact with metal ions in the DNA cleavage/ligation active site of topoisomerase IIalpha. Furthermore, we propose that M(1)(2+) interacts with E461, D543, and D545 and M(2)(2+) interacts with E461 and D541.

Author information

Author/s: Deweese, Joseph E (JE); Guengerich, F Peter (FP); Burgin, Alex B (AB); Osheroff, Neil (N);

Affiliation: Department of Biochemistry, Vanderbilt University School of Medicine, Nashville, Tennessee 37232-0146, USA.

Grants: ES010375 (Agency:NIEHS NIH HHS) ; GM033944 (Agency:NIGMS NIH HHS) ; GM053960 (Agency:NIGMS NIH HHS) ; T32 CA09592 (Agency:NCI NIH HHS)

Journal and publication information

Publication Type: In Vitro; Journal Article; Research Support, N.I.H., Extramural

Journal: Biochemistry (Biochemistry), published in United States. (Language: eng)

Reference: 2009-Sep; vol 48 (issue 38) : pp 8940-7

Dates: Created 2009/09/22; Completed 2009/10/09;

PMID: 19697956, status: MEDLINE (last retrieval date: 10/9/2009, IMS Date: )

Sourced from the National Library of Medicine. Abstract text and other information may be subject to copyright.

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MeSH headings (categories)

This article was linked to the MESH Headings shown below.

Amino Acid Sequence Amino Acid Substitution Antigens, Neoplasm - chemistry; genetics; metabolism Base Sequence Catalytic Domain - genetics Cations, Divalent - metabolism Conserved Sequence DNA - genetics; metabolism DNA Primers - genetics DNA Topoisomerases, Type II, Eukaryotic - chemistry; genetics; metabolism DNA-Binding Proteins - chemistry; genetics; metabolism Humans

Humans Kinetics Metals - metabolism Models, Chemical Molecular Sequence Data Mutagenesis, Site-Directed Nucleic Acid Conformation Oligodeoxyribonucleotides - genetics; metabolism Plasmids - chemistry; metabolism Recombinant Proteins - chemistry; genetics; metabolism Sequence Homology, Amino Acid Substrate Specificity

Associated Chemicals: Antigens, Neoplasm (0) ; Cations, Divalent (0) ; DNA Primers (0) ; DNA-Binding Proteins (0) ; Metals (0) ; Oligodeoxyribonucleotides (0) ; Recombinant Proteins (0) ; DNA (9007-49-2) ; DNA Topoisomerases, Type II, Eukaryotic (EC 5.99.1.-) ; DNA topoisomerase II alpha (EC 5.99.1.3)

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