Importance of the fourth alpha-helix within the CAP homology domain of type II topoisomerase for DNA cleavage site recognition and quinolone action.
We report that point mutations causing alteration of the fourth alpha-helix (alpha4-helix) of the CAP homology domain of eukaryotic (Saccharomyces cerevisiae) type II topoisomerases (Ser(740)Trp, Gln(743)Pro, and Thr(744)Pro) change the selection of type II topoisomerase-mediated DNA cleavage sites promoted by Ca(2+) or produced by etoposide, the fluoroquinolone CP-115,953, or mitoxantrone. By contrast, Thr(744)Ala substitution had minimal effect on Ca(2+)- and drug-stimulated DNA cleavage sites, indicating the selectivity of single amino acid substitutions within the alpha4-helix on type II topoisomerase-mediated DNA cleavage. The equivalent mutation in the gene for Escherichia coli gyrase causing Ser(83)Trp also changed the DNA cleavage pattern generated by Ca(2+) or quinolones. Finally, Thr(744)Pro substitution in the yeast type II topoisomerase rendered the enzyme sensitive to antibacterial quinolones. This study shows that the alpha4-helix within the conserved CAP homology domain of type II topoisomerases is critical for selecting the sites of DNA cleavage. It also demonstrates that selective amino acid residues in the alpha4-helix are important in determining the activity and possibly the binding of quinolones to the topoisomerase II-DNA complexes.
Strumberg
Dirk
D
Department of Internal Medicine and Medical Oncology, West German Cancer Center, University Medical School of Essen, 45122 Essen, Germany. dirk.strumberg@uni-essen.de
Nitiss
John L
JL
Dong
Jiaowang
J
Walker
Jerrylaine
J
Nicklaus
Marc C
MC
Kohn
Kurt W
KW
Heddle
Jonathan G
JG
Maxwell
Anthony
A
Seeber
Siegfried
S
Pommier
Yves
Y
D042462:4-Quinolones; D000595:Amino Acid Sequence; D000890:Anti-Infective Agents / Q000494:pharmacology*; D002118:Calcium / Q000494:pharmacology; D053938:DNA Fragmentation; D027081:DNA Gyrase / Q000378:metabolism; D004250:DNA Topoisomerases, Type II / Q000096:biosynthesis / Q000378:metabolism*; D004269:DNA, Bacterial / Q000737:chemistry / Q000378:metabolism*; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000201:enzymology; D024841:Fluoroquinolones; D011487:Protein Conformation; D039381:RNA Cap-Binding Proteins / Q000378:metabolism*; D020133:Reverse Transcriptase Polymerase Chain Reaction; D012441:Saccharomyces cerevisiae / Q000201:enzymology; D012689:Sequence Homology, Nucleic Acid; D059005:Topoisomerase II Inhibitors
D042462:4-Quinolones; D000890:Anti-Infective Agents; D004269:DNA, Bacterial; D024841:Fluoroquinolones; D039381:RNA Cap-Binding Proteins; D059005:Topoisomerase II Inhibitors; C070116:CP 115953; D027081:DNA Gyrase; D004250:DNA Topoisomerases, Type II; D002118:Calcium