Inhibition of Flp Recombinase by the Topoisomerase I-targeting Drugs, Camptothecin and NSC-314622

Abstract

Recombinases of the -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. 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). In the first step of recombination, an active site tyrosine nucleophile attacks the target phosphodiester bond in DNA to generate a 3Ј-phosphotyrosyl linkage and a free 5Ј-hydroxyl group. In the second step leading to strand rejoining, the 5Ј-hydroxyl group is the nucleophile, and the 3Ј-phosphotyrosyl bond is its target.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). However, the two ligation modes are not mutually exclusive, as evident from the fact that some Int recombinases can relax supercoiled DNA in vitro under certain conditions while some type IB topoisomerases can mediate recombination and resolution of Holliday junctions (4 -8). Recent structural data have further consolidated the relationship between the Int family recombinases and the type IB topoisomerases. Despite the lack of overall sequence homology between the two enzyme groups, the tertiary folds within the catalytic domains are strikingly similar between the Int-type recombinases, including Flp (9) and Cre (10), and eukaryotic topoisomerase I (topo I) (11)(12)(13)(14). Moreover, the critical catalytic moieties include two nearly identical tetrads, RHRH/W in the recombinases and RKRH in the topoisomerases, together with the invariant tyrosine nucleophile (1,11).In this study we have further probed the functional relationship between the Int family recombinase and type IB topoisomerase active sites by inves...