Cell, Vol. 22, 269-276, November 1980 (Part l), Copyright 0 1960 by MIT E. coli and M. luteus DNA Topoisomerase I Can Catalyze Catenation or Decatenation of Double- Stranded DNA Rings Yuk-thing Tse and James C. Wang Department of Biochemistry and Molecular Biology Harvard University Cambridge, Massachusetts 02138 Summary Escherichia coli and Micrococcus luteus DNA to- poisomerase I are found to promote catenation of double-stranded DNA rings. At low DNA concentra- tion dimeric catenanes are the major catenated products; at high DNA concentration or when sper- midine is present, catenanes containing more than two rings are formed. There is no requirement of extensive sequence homology between the com- ponent rings forming a catenane; dimeric catenanes between Pseudomonas phage PM2 DNA and E. coli plasmid pBR322 are readily formed. The formation of a dimeric catenane by these type I topoisomer- ases, however, requires the presence of at least one preexisting single-chain scission in one of the two component rings. This is in contrast to the cases with the type II DNA topoisomerases which can form catenanes made of covalently closed rings only. The catenanes formed by the type I enzymes can be unlinked by the same enzymes, or by DNA gyrase, a type II enzyme, upon dilution of the iso- lated catenanes. The catenation and decatenation of duplex DNA rings adds a fourth type of reaction promoted by these type I DNA topoisomerases to the three reported previously: relaxation of super- helical DNA, interconversion between single- stranded DNA rings with and without knots and the intertwining of single-stranded DNA rings of com- plementary sequences into a covalently closed du- plex ring with a high linking number. All four topo- isomerization reactions involve the crossing of one DNA strand through a transient break of another DNA strand. The new reaction reported here sug- gests that such a crossover event might not require pairing of complementary nucleotide sequences. Introduction DNA topoisomerases are enzymes that catalyze the concerted breaking and rejoining of DNA backbone bonds (for reviews see Champoux, 1978; Wang and Liu, 1979; Cozzarelli, 1980). Recent studies suggest that these enzymes can be classified into two cate- gories. For the type I enzymes the breaking and rejoining events occur in one strand at a time and appear to be uncoordinated; for the type II enzymes the breaking and rejoining of both strands appear to occur in a coordinated fashion (Brown and Cozzarelli, 1979; Liu, Liu and Alberts, 1979, 1980; Hsieh and Brutlag, 1980; Mizuuchi et al., 1980). Three types of DNA topoisomerization catalyzed by the type I en- zymes have been reported to date: the removal of superhelical turns, the linking of single-stranded rings of complimentary sequences and the interconversion between single-stranded rings with and without knots (reviewed by Wang and Liu, 1979). The type II en- zymes also catalyze three types of reactions: the removal or introduction of superhelical turns, the link- ing of double-stranded rings with or without gross sequence homology and the interconversion between double-stranded rings with and without knots (Gellert et al., 1976; Liu and Wang, 1978; Sugino et al., 1978; Liu et al., 1979, 1980; Hsieh and Brutlag, 1980; Kreuzer and Cozzarelli, 1980; Baldi et al., 1980; Mizuuchi et al., 1980). Although both type I and type II enzymes catalyze the alteration of the linking num- ber of a covalently closed DNA, it appears that in a type I enzyme-catalyzed reaction the linking number of a DNA can change by one at a time, whereas in a type II enzyme-catalyzed reaction the linking number is altered by multiples of two (Pulleybank et al., 1975; Brown and Cozzarelli, 1979; Hsieh and Brutlag, 1980; Liu et al., 1980; Mizuuchi et al., 1980). Topologically, it is expected that the breakage of a duplex DNA ring, followed by the passage of a segment of the DNA through the break and finally the resealing of the double-stranded break, changes the linking number of the DNA by two, if in the broken state the ends are not allowed to rotate relative to each other around the helix axis (Fuller, 1978). Thus all the differences ob- served between the type I and type II enzymes cata- lyzed reactions are entirely consistent with the basis of their classification. During the course of our studies on E. coli and M. luteus DNA topoisomerase I we have observed that these type I enzymes also promote the linking of duplex rings into catenanes. This is surprising in view of the distinction made above between type I and type II enzymes. We have therefore examined this reaction in some detail. In this communication we present first our results that authentic catenanes consisting of double-strand rings (Figure la), rather than fused rings of the types shown below (Figures 1 b and 1 c), are formed in this novel reaction. We then show that in the formation of a dimeric catenane by such en- zymes at least one of the two component rings must contain one or more single-chain scissions and that no gross nucleotide sequence homology between the two component rings is required. Finally we discuss the implications of these results in relation to the mechanisms of DNA topoisomerization. Results E. coli and M. luteus DNA Topoisomerase I Can Catalyze the Formation of Catenanes between Double-Stranded DNA Rings Figure 2 depicts the electrophoretic patterns, in aga- rose gel containing ethidium, of a PM2 DNA sample before and after incubation with M. luteus DNA topo- isomerase I. For the particular DNA sample used,