Mutation of cysteine residue 455 to alanine in human topoisomerase IIalpha confers hypersensitivity to quinones: enhancing DNA scission by closing the N-terminal protein gate.
Several quinone-based metabolites of industrial and environmental toxins are potent topoisomerase II poisons. These compounds act by adducting the protein, and previous studies suggest that they increase levels of enzyme-associated DNA strand breaks by at least two potential mechanisms. Quinones act directly on the DNA cleavage-ligation equilibrium of topoisomerase II by inhibiting the rate of ligation. They also block the N-terminal gate of the protein, thereby stabilizing topoisomerase II in its "closed clamp" form and trapping DNA in the central annulus of the enzyme. It has been proposed that this latter activity enhances DNA cleavage by increasing the population of enzyme molecules with DNA in their active sites, but a causal relationship has not been established. In order to more fully characterize the mechanistic basis for quinone action against topoisomerase II, the present study characterized the sensitivity of human topoisomerase IIalpha carrying a Cys455-->Ala mutation (top2alphaC455A) toward quinones. Cys455 was identified as a site of quinone adduction by mass spectrometry. The mutant enzyme was approximately 1.5-2-fold hypersensitive to 1,4-benzoquinone and the polychlorinated biphenyl quinone 4'Cl-2,5pQ, but it displayed wild-type sensitivity to traditional topoisomerase II poisons. The ability of 1,4-benzoquinone to inhibit DNA ligation mediated by top2alphaC455A was similar to that of wild-type topoisomerase IIalpha. However, the quinone induced approximately 3 times the level of clamp closure with the mutant enzyme. These findings strongly support the hypothesis that the ability of quinones to block the N-terminal gate of the type II enzyme contributes to their actions as topoisomerase II poisons.
Bender
Ryan P
RP
Department of Biochemistry, Vanderbilt University School of Medicine, Nashville, Tennessee 37232-0146, USA.
Osheroff
Neil
N
D000409:Alanine / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D019943:Amino Acid Substitution; D000951:Antigens, Neoplasm / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D016227:Benzoquinones / Q000737:chemistry / Q000494:pharmacology; D003545:Cysteine / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D053837:DNA Cleavage / Q000187:drug effects*; D004250:DNA Topoisomerases, Type II / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D004278:DNA, Superhelical / Q000235:genetics / Q000378:metabolism; D004268:DNA-Binding Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D005047:Etoposide / Q000737:chemistry / Q000494:pharmacology; D006801:Humans; D007700:Kinetics; D013058:Mass Spectrometry; D008958:Models, Molecular; D050505:Mutant Proteins / Q000737:chemistry / Q000378:metabolism; D009154:Mutation; D017434:Protein Structure, Tertiary; D011809:Quinones / Q000737:chemistry / Q000494:pharmacology*; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D013696:Temperature
D000951:Antigens, Neoplasm; D016227:Benzoquinones; D004278:DNA, Superhelical; D004268:DNA-Binding Proteins; D050505:Mutant Proteins; D011809:Quinones; D011994:Recombinant Proteins; C004532:quinone; D005047:Etoposide; D004250:DNA Topoisomerases, Type II; D003545:Cysteine; D000409:Alanine