Characterization of an altered DNA catalysis of a camptothecin-resistant eukaryotic topoisomerase I.
Nucleic Acids Res
; 21(3): 593-600, 1993 Feb 11.
Article
in En
| MEDLINE
| ID: mdl-8382801
ABSTRACT
We investigated topoisomerase I activity at a specific camptothecin-enhanced cleavage site by use of a partly double-stranded DNA substrate. The cleavage site belongs to a group of DNA topoisomerase I sites which is only efficiently cleaved by wild-type topoisomerase I (topo I-wt) in the presence of camptothecin. With a mutated camptothecin-resistant form of topoisomerase I (topo I-K5) previous attempts to reveal cleavage activity at this site have failed. On this basis it was questioned whether the mutant enzyme has an altered DNA sequence recognition or a changed rate of catalysis at the site. Utilizing a newly developed assay system we demonstrate that topo I-K5 not only recognizes and binds to the strongly camptothecin-enhanced cleavage site but also has considerable cleavage/religation activity at this particular DNA site. Thus, topo I-K5 has a 10-fold higher rate of catalysis and a 10-fold higher affinity for DNA relative to topo I-wt. Our data indicate that the higher cleavage/religation activity of topo I-K5 is a result of improved DNA binding and a concomitant shift in the equilibrium between cleavage and religation towards the religation step. Thus, a recently identified point mutation which characterizes the camptothecin-resistant topo I-K5 has altered the enzymatic catalysis without disturbing the DNA sequence specificity of the enzyme.
Full text:
1
Collection:
01-internacional
Database:
MEDLINE
Main subject:
Camptothecin
/
DNA
/
DNA Topoisomerases, Type I
Type of study:
Prognostic_studies
Limits:
Humans
Language:
En
Journal:
Nucleic Acids Res
Year:
1993
Document type:
Article
Affiliation country:
Denmark