Mutation of a Conserved Serine Residue in a Quinolone-resistant Type II Topoisomerase Alters the Enzyme-DNA and Drug Interactions

Abstract

A Ser740 3 Trp mutation in yeast topoisomerase II (top2) and of the equivalent Ser 83 in gyrase results in resistance to quinolones and confers hypersensitivity to etoposide (VP-16). We characterized the cleavage complexes induced by the top2 S740W in the human c-myc gene. In addition to resistance to the fluoroquinolone CP-115,953, top2 S740W induced novel DNA cleavage sites in the presence of VP-16, azatoxin, amsacrine, and mitoxantrone. Analysis of the VP-16 sites indicated that the changes in the cleavage pattern were reflected by alterations in base preference. C at position ؊2 and G at position ؉6 were observed for the top2 S740W in addition to the previously reported C؊1 and G؉5 for the wildtype top2. The VP-16-induced top2 S740W cleavage complexes were also more stable. The most stable sites had strong preference for C؊1, whereas the most reversible sites showed no base preference at positions ؊1 or ؊2. Different patterns of DNA cleavage were also observed in the absence of drug and in the presence of calcium. These results indicate that the Ser 740 3 Trp mutation alters the DNA recognition of top2, enhances its DNA binding, and markedly affects its interactions with inhibitors. Thus, residue 740 of top2 appears critical for both DNA and drug interactions.DNA topoisomerases are enzymes that catalyze changes in the topology of DNA via a mechanism involving the transient breakage and rejoining of phosphodiester bonds in the DNA backbone (1). Studies in both prokaryotic and eukaryotic cells have demonstrated the importance of topoisomerases in transcription, DNA replication, and chromosome segregation. The type II topoisomerases, which make transient double-strand breaks and change the linking number of DNA in steps of two, play key roles in chromosome structure. In eukaryotic cells, these enzymes are essential for chromosome condensation/decondensation and decatenation of chromosome loops during mitosis (2, 3).Topoisomerase II (top2) 1 has also been identified as a major target of chemotherapeutic agents that are specifically active against prokaryotic (4) or cancer cells (5-8). Fluoroquinolones are antibacterial agents that target DNA gyrase (the prokaryotic type II topoisomerase) (9), whereas a variety of DNAintercalating agents such as anthracyclines, amsacrine, and mitoxantrone and nonintercalating agents such as the epipodophyllotoxin etoposide (VP-16) are active against eukaryotic top2 and are clinically important anti-cancer agents (5-8).Azatoxin is also a nonintercalative top2 poison (10).Recently, it has been shown that 6,8-difluoro-7-(4Ј-hydroxyphenyl)-1-cyclopropyl-4-quinolone-3-carboxylic acid (CP-115, 953), a fluoroquinolone closely related to ciprofloxacin, is highly toxic to mammalian cells in culture (11,12). Studies in yeast demonstrated that top2 is the primary physiological target for this quinolone (13). Unlike etoposide, which stabilizes top2-mediated DNA cleavage primarily by inhibiting the religation reaction of top2, CP-115,953 stabilizes DNA cleavage by enhancing the forward rate o...

Molecular Analysis of Yeast and Human Type II Topoisomerases

“…However, the stability was considerably less than for the human top2␣ S763W protein. As already shown for yeast top2 S740W (24), cleavage sites with slow reversibility exhibited highly significant preferences for C Ϫ1 in combination with less strong C Ϫ2 preference in human top2␣ S763W , whereas rapidly reversible cleavage sites did not show any preferences at positions Ϫ1 and Ϫ2 (data not shown). Hence, both mutations Ser 740 3 Trp in yeast and Ser 763 3 Trp in human top2␣ similarly alter the DNA recognition of the corresponding enzyme, markedly affect the interaction with inhibitors, and enhance the stability of the top2 cleavage complexes in the presence of etoposide.…”

“…Reactions were stopped by adding 1% SDS (v/v) and further digested with proteinase K (0.4 mg/ml final concentration for 30 min at 55°C). Calcium-promoted DNA cleavage was performed in the same buffer with 5 mM CaCl 2 instead of MgCl 2 (24).…”

“…Since the Ser 740 3 Trp mutation in yeast affects the DNA cleavage patterns induced by both intercalating and non-intercalating drugs (24) and confers partial resistance to fluoroquinolones and collateral hypersensitivity to etoposide (38), we compared the drug-induced DNA cleavage sites for the human …”

“…In the case of etoposide, no significant difference in base preference was observed despite clear differences in observed DNA cleavage patterns. (24). To analyze the effect of the homologous mutation in human top2␣ (Ser 763 3 Trp), we compared the calcium-promoted DNA cleavages for both mutant proteins (Fig.…”

“…In this way, a clear and detailed comparison between yeast and human top2 is warranted and necessary. Since mutation of a conserved serine residue (Ser 740 3 Trp) in yeast top2 was recently reported to alter both enzyme-DNA and drug interactions (24), the homologous mutation (Ser 763 3 Trp) in human top2␣ was analyzed in this study. Preparation of End-labeled DNA Fragments by PCR-Three sets of labeled DNA fragments were prepared from the human c-MYC gene by PCR.…”