Abstract
Recombinases of the lambda-Int family and type IB topoisomerases act by introducing transient single strand breaks in DNA using chemically identical reaction schemes. Recent structural data have supported the relationship between the two enzyme groups by revealing considerable similarities in the architecture of their catalytic pockets. In this study we show that the Int-type recombinase Flp is inhibited by the two structurally unrelated topoisomerase I-directed anti-cancer drugs, camptothecin (CPT) and NSC-314622. The interaction of these drugs with topoisomerase I is very specific with several single amino acid substitutions conferring drug resistance to the enzyme. Thus, the observed interaction of CPT and NSC-314622 with Flp, which is comparable to their interaction with the cleavage complex formed by topoisomerase I, strongly supports a close mechanistic and evolutionary relationship between the two enzymes. The results suggest that Flp and other Int family recombinases may provide model systems for dissecting the molecular mechanisms of topoisomerase I-directed anti-cancer therapeutic agents.
Highlights
Members of the integrase (Int)1 family of site-specific recombinases (-integrase, P1 Cre recombinase, Escherichia coli XerC/XerD recombinase, Saccharomyces cerevisiae Flp, and Zygosaccharomyces rouxii R recombinases among several others) all carry out conservative site-specific recombination using a basic type IB topoisomerase reaction scheme (1, 2)
The nucleophilic attack is directed across partner substrates so that strand ligation occurs in the recombinant configuration, which is opposed to the typical type IB topoisomerase reaction in which ligation restores the original phosphodiester bond (3)
In the present study we have demonstrated that CPT and NSC-314622 inhibit Flp recombinase by blocking the active site from the incoming nucleophile for strand cleavage or ligation
Summary
Integrase; CPT, camptothecin; NSC-314622, 5,6-dihydro-5,11-diketo-2,3-dimethoxy-6-methyl-8,9methyllenedioxy-11H-indenol(1,2-c)isoquinoline; topo I, topoisomerase I. NSC-314622 is structurally unrelated to the camptothecins, its mode of action appears to be similar to that of CPT (17) Both drugs inhibit the religation step of topo I catalysis, whereas they have no effect on the cleavage reaction (16 –19). It is well established that the drugs do not interact with either topo I or DNA separately (19) Rather, they form a ternary complex with the cleavage intermediate, blocking the active site of the covalently bound enzyme (18, 20). In the present study we demonstrate that the Int type recombinase, the Flp protein, is inhibited by CPT and NSC-314622 This is the first report of an enzyme other than eukaryotic topo I being sensitive toward these drugs. The results demonstrate several similarities and subtle differences between Flp and topo I in the mode of inhibition by the two drugs and suggest that the simple members of the Int family such as Flp and Cre may be exploited to dissect the molecular action of the topo I-directed anti-cancer drugs
Sign-in/Register to view highlights & summary Sign-in/Register to access full text options 
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a callDisclaimer: All third-party content on this website/platform is and will remain the property of their respective owners and is provided on "as is" basis without any warranties, express or implied. Use of third-party content does not indicate any affiliation, sponsorship with or endorsement by them. Any references to third-party content is to identify the corresponding services and shall be considered fair use under The CopyrightLaw.
