A catalytic domain of eukaryotic DNA topoisomerase I.
Eukaryotic type IB topoisomerases catalyze the cleavage and rejoining of DNA strands through a DNA-(3'-phosphotyrosyl)-enzyme intermediate. The 314-amino acid vaccinia topoisomerase is the smallest member of this family and is distinguished from its cellular counterparts by its specificity for cleavage at the target sequence 5'-CCCTT downward arrow. Here we show that Topo-(81-314), a truncated derivative that lacks the N-terminal domain, performs the same repertoire of reactions as the full-sized topoisomerase: relaxation of supercoiled DNA, site-specific DNA transesterification, and DNA strand transfer. Elimination of the N-terminal domain slows the rate of single-turnover DNA cleavage by 10(-3.6), but has little effect on the rate of single-turnover DNA religation. DNA relaxation and strand cleavage by Topo-(81-314) are inhibited by salt and magnesium; these effects are indicative of reduced affinity in noncovalent DNA binding. We report that identical properties are displayed by a full-length mutant protein, Topo(Y70A/Y72A), which lacks two tyrosine side chains within the N-terminal domain that contact the DNA target site in the major groove. We speculate that Topo-(81-314) is fully competent for transesterification chemistry, but is compromised with respect to a rate-limiting precleavage conformational step that is contingent on DNA contacts made by Tyr-70 and Tyr-72.
Cheng
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Molecular Biology Program, Sloan-Kettering Institute, New York, New York 10021, USA.
Shuman
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D001665:Binding Sites; D002918:Chymotrypsin / Q000494:pharmacology; D004264:DNA Topoisomerases, Type I / Q000378:metabolism / Q000648:ultrastructure*; D004278:DNA, Superhelical / Q000378:metabolism*; D004268:DNA-Binding Proteins / Q000737:chemistry*; D007700:Kinetics; D008274:Magnesium / Q000494:pharmacology; D009994:Osmolar Concentration; D011994:Recombinant Proteins; D017384:Sequence Deletion; D013329:Structure-Activity Relationship; D014443:Tyrosine / Q000737:chemistry; D014616:Vaccinia virus / Q000201:enzymology*
D004278:DNA, Superhelical; D004268:DNA-Binding Proteins; D011994:Recombinant Proteins; D014443:Tyrosine; D002918:Chymotrypsin; D004264:DNA Topoisomerases, Type I; D008274:Magnesium