DNA Duplexes Containing 3′-Deoxynucleotides as Substrates for DNA Topoisomerase I Cleavage and Ligation

Abstract

The DNA cleavage-ligation reaction of DNA topoisomerase I was investigated employing synthetic DNA substrates containing 3′-deoxyadenosine or 3′-deoxythymidine at specific sites and acceptor oligonucleotides of different lengths. The modified nucleotides were substituted systematically within the putative enzyme-binding domain and also next to the high efficiency cleavage site to determine the effect of single base changes on enzyme function. Depending on the site of substitution, the facility of the cleavage and ligation reactions were altered. The bases at positions −1 and −2 on the noncleaved strand were found to be important for determining the site of cleavage. Inclusion of 3′-deoxythymidine in the scissile strand at position −1 permitted the demonstration that topoisomerase I can cleave and form a 2′ → 5′-phosphodiester linkage. Partial duplexes doubly modified at positions −4 or −6 in the noncleaved strand and at positions +1 or −1 within scissile strand were not good substrates for topoisomerase I, showing that cleavage can depend importantly on binding interactions based on structural alterations at spatially separated sites. Substitution of a 3′-deoxynucleotide on the scissile strand at position −6 enhanced formation of the ligation product resulting from cleavage at site 1 and suppressed cleavage at site 2. The DNA cleavage-ligation reaction of DNA topoisomerase I was investigated employing synthetic DNA substrates containing 3′-deoxyadenosine or 3′-deoxythymidine at specific sites and acceptor oligonucleotides of different lengths. The modified nucleotides were substituted systematically within the putative enzyme-binding domain and also next to the high efficiency cleavage site to determine the effect of single base changes on enzyme function. Depending on the site of substitution, the facility of the cleavage and ligation reactions were altered. The bases at positions −1 and −2 on the noncleaved strand were found to be important for determining the site of cleavage. Inclusion of 3′-deoxythymidine in the scissile strand at position −1 permitted the demonstration that topoisomerase I can cleave and form a 2′ → 5′-phosphodiester linkage. Partial duplexes doubly modified at positions −4 or −6 in the noncleaved strand and at positions +1 or −1 within scissile strand were not good substrates for topoisomerase I, showing that cleavage can depend importantly on binding interactions based on structural alterations at spatially separated sites. Substitution of a 3′-deoxynucleotide on the scissile strand at position −6 enhanced formation of the ligation product resulting from cleavage at site 1 and suppressed cleavage at site 2. The DNA topoisomerases alter DNA topology via the introduction of transient breaks in the phosphodiester backbone of this biopolymer (1Chen A.Y. Liu L.F. Annu. Rev. Pharmacol. Toxicol. 1994; 34: 191-218Crossref PubMed Scopus (780) Google Scholar,2Gupta M. Fujimori A. Pommier Y. Biochim. Biophys. Acta. 1995; 1262: 1-14Crossref PubMed Scopus (284) Google Scholar). These enzymes participate in the essential cellular processes of DNA replication, transcription, and recombination (3Froelich-Ammon S.J. Osheroff N. J. Biol. Chem. 1995; 270: 21429-21432Abstract Full Text Full Text PDF PubMed Scopus (509) Google Scholar). DNA topoisomerases are classified into two groups based on their mode of DNA strand scission: the type I enzymes mediate the transient single-strand breakage of a DNA substrate, whereas the type II topoisomerases break both strands. Because of their essential role in the cell, the eukaryotic topoisomerases have become important targets for the development of antitumor agents (4Kingsbury W.D. Boehm J.C. Jakas D.R. Holden K.G. Hecht S.M. Gallagher G. Caranfa M.J. McCabe F.L. Faucette L.F. Johnson R.K. Hertzberg R.P. J. Med. Chem. 1991; 34: 98-107Crossref PubMed Scopus (543) Google Scholar). The mechanism of DNA cleavage involves nucleophilic attack of a tyrosine OH group in the active site of topoisomerase I on the phosphate ester backbone, resulting in the attachment of tyrosine to the DNA through an oligonucleotide 3′-phosphate with concomitant release of an oligonucleotide having a free 5′-OH terminus (5Champoux J.J. J. Biol. Chem. 1981; 256: 4805-4809Abstract Full Text PDF PubMed Google Scholar). After strand passage of the free DNA strand around the unbroken strand, religation of the broken strand occurs by a process believed to involve reversal of the cleavage reaction. Under normal circumstances the cleavage and ligation reactions are tightly coupled with low steady-state concentrations of the covalent intermediate (6Champoux J.J. Proc. Natl. Acad. Sci. U. S. A. 1976; 73: 3488-3491Crossref PubMed Scopus (86) Google Scholar). However, the two partial reactions can be uncoupled in vitro, e.g. by site-specific cleavage of partially double-stranded substrates containing a high efficiency cleavage site (see Fig. 1) (7Svejstrup J.Q. Christiansen K. Gromova I.I. Andersen A.H. Westergaard O. J. Mol. Biol. 1991; 222: 669-678Crossref PubMed Scopus (153) Google Scholar). Cleavage of the “suicide substrate” occurs without sequential religation due to the instability of the duplex involving the truncated strand downstream from the site of cleavage; loss of this oligonucleotide traps the topoisomerase I-DNA covalent intermediate. The covalently bound enzyme is catalytically competent, as may be judged by formation of the intact duplex upon admixture of an oligonucleotide complementary to the single-stranded region in the enzyme-DNA covalent binary complex (see Fig. 1 B). The covalent binary complex also undergoes ligation with partially complementary acceptors containing a free 5′-OH group (8McCoubrey Jr., W.K. Champoux J.J. J. Biol. Chem. 1986; 261: 5130-5137Abstract Full Text PDF PubMed Google Scholar). The topoisomerases I from all species characterized thus far recognize specific nucleotide sequences in their DNA substrates, which are cleaved with high efficiency (see Fig. 1) (9Svejstrup J.Q. Christiansen K. Andersen A.H. Lund K. Westergaard O. J. Biol. Chem. 1990; 265: 12529-12535Abstract Full Text PDF PubMed Google Scholar, 10Christiansen K. Svejstrup A.B.D. Andersen A.H. Westergaard O. J. Biol. Chem. 1993; 268: 9690-9701Abstract Full Text PDF PubMed Google Scholar). Such sites can be identified by footprinting analysis (11Stevnsner T. Mortensen U.H. Westergaard O. Bonven B.J. J. Biol. Chem. 1989; 264: 10110-10113Abstract Full Text PDF PubMed Google Scholar). Although the cleavage-ligation reactions have been studied in some detail, further investigation is needed to define the possible roles of single nucleotides within high efficiency sequences on enzyme binding, as well as cleavage and ligation (12Vosberg H.P. Curr. Top. Microbiol. Immunol. 1985; 114: 19-102PubMed Google Scholar). Different approaches have been utilized to determine the role of individual nucleotides, including phosphate ethylation (13Krogh S. Mortensen U.H. Westergaard O. Bonven B.J. Nucleic Acids Res. 1991; 19: 1235-1241Crossref PubMed Scopus (60) Google Scholar) and deoxyguanosine N7-methylation and subsequent depurination to create abasic sites (11Stevnsner T. Mortensen U.H. Westergaard O. Bonven B.J. J. Biol. Chem. 1989; 264: 10110-10113Abstract Full Text PDF PubMed Google Scholar). Abasic sites within the scissile strand on the 5′-side of the high efficiency cleavage site rendered the DNA substrate refractory to cleavage by topoisomerase I (11Stevnsner T. Mortensen U.H. Westergaard O. Bonven B.J. J. Biol. Chem. 1989; 264: 10110-10113Abstract Full Text PDF PubMed Google Scholar). In addition, Pourquier et al. (14Pourquier P. Ueng L.-M. Kohlhagen G. Mazumder A. Gupta M. Kohn K.W. Pommier Y. J. Biol. Chem. 1997; 272: 7792-7796Abstract Full Text Full Text PDF PubMed Scopus (194) Google Scholar) have incorporated deoxyuridine and abasic sites into the noncleaved strand of a DNA substrate and investigated the effect on topoisomerase I-mediated cleavage. They found that the position of cleavage was altered and that the new site of cleavage was dependent on the location of the site modified. We have reported previously that topoisomerase I can catalyze the ligation to the covalent topoisomerase I-DNA binary complex of complementary acceptor oligonucleotides having modified nucleophiles at the 5′ terminus; the resulting duplexes have altered connectivity at the site of ligation (15Henningfeld K.A. Arslan T. Hecht S.M. J. Am. Chem. Soc. 1996; 118: 1701-1714Crossref Scopus (34) Google Scholar). Recently, we have extended these findings by employing a modified acceptor oligonucleotide having the 5′-terminal deoxyadenosine linked to the remainder of the oligomer through a 2 → 5′-phosphodiester bond (see Fig. 1C). When employed in the presence of a partial DNA duplex that afforded a single-stranded region complementary to the acceptor upon cleavage by topoisomerase I, the modified oligonucleotide was incorporated into a newly formed duplex (16Arslan T. Abraham A.T. Hecht S.M. Nucleosides Nucleotides. 1998; 17: 515-530Crossref PubMed Scopus (12) Google Scholar). The extent of (modified) duplex formation was only ∼10% of that achieved with the respective unmodified acceptor oligonucleotide after 1 h, but the yield of the modified duplex could be increased to 20% after a 13-h incubation (16Arslan T. Abraham A.T. Hecht S.M. Nucleosides Nucleotides. 1998; 17: 515-530Crossref PubMed Scopus (12) Google Scholar). In addition, modified partial duplexes were utilized as substrates; a single 3′-deoxyadenosine within the DNA topoisomerase region on the scissile The facility of cleavage or ligation was altered in a dependent on the location of the 3′-deoxyadenosine further the role of single nucleotides within the domain on substrate and cleavage by topoisomerase I, partial duplex substrates containing in the scissile and noncleaved strand have been we that on the scissile or noncleaved can the topoisomerase I-mediated cleavage and ligation of modified nucleotides within the substrate can cleavage and ligation topoisomerase I can effect the cleavage and ligation of a 2′ → ester within the DNA substrate, and alterations in efficiency of enzyme by the introduction of a 3′-deoxynucleotide at a specific site can be by introduction of the complementary 3′-deoxynucleotide at the site on the and were from U. S. was from and the for oligonucleotide were from were from and was from was on a was on 20% in containing 1 were by at with and a with DNA analysis was by of the PubMed Scopus Google Scholar) for single-stranded from a was for all oligonucleotides were from or on a DNA 1985; PubMed Scopus Google Scholar). The oligonucleotides on the DNA were and cleaved from the by with at for The were by The was with of and with of oligonucleotides in of were the The was with of The oligonucleotides were with of and with of of the oligonucleotides was with of containing oligonucleotides were on 20% the DNA was by and and by The DNA was with J. T. Scholar). were in of containing and The was to for and to a of to the low DNA oligonucleotide of the strand and a of the to of the The partial duplex was with topoisomerase I Johnson R.K. Hecht S.M. Chem. Res. Toxicol. 1993; PubMed Scopus Google Scholar) in the presence of a of or acceptor oligonucleotide in a reaction containing and 1 The reaction were at for by with 1 for 1 The reaction were in of and The reaction were at for and on were to a 20% and at for in containing 1 of of containing 1 of duplex DNA and 2 of The reactions were by the of of After the reactions were by the of 2 of and of The reactions were at for and on was to a 20% The of the were with the resulting from and reactions PubMed Scopus Google Scholar). The 3′-deoxynucleotide were to (16Arslan T. Abraham A.T. Hecht S.M. Nucleosides Nucleotides. 1998; 17: 515-530Crossref PubMed Scopus (12) Google Scholar, Scopus Google Scholar) and incorporated into the DNA substrates within the topoisomerase region on the scissile and noncleaved for 1985; PubMed Scopus Google Scholar). Cleavage from the and of the base and phosphate groups was as oligonucleotides were by and by 20% The formation of modified duplexes was by which the position of the modified nucleotide in of the partial duplexes (15Henningfeld K.A. Arslan T. Hecht S.M. J. Am. Chem. Soc. 1996; 118: 1701-1714Crossref Scopus (34) Google Scholar) not The topoisomerase I-mediated cleavage and ligation reactions were investigated partial duplexes containing high efficiency topoisomerase I cleavage sequences K. Bonven B.J. Westergaard O. J. Mol. Biol. PubMed Scopus Google Scholar) and a single within the noncleaved strand at The and acceptor oligonucleotides were complementary to of the duplex that single-stranded upon cleavage at sites 1 and We investigated the topoisomerase I-mediated cleavage-ligation reaction partial duplexes modified within the noncleaved strand at positions or The partial duplexes containing these were with DNA topoisomerase I in the presence of the acceptor oligonucleotide at for The reaction were by with and with to the covalently bound The reactions were on a 20% in the topoisomerase I-mediated cleavage and ligation in the presence the acceptor oligomer afforded the noncleaved strand was modified at positions −4 or at position −1 or −2 in the noncleaved strand ligation with the the presence of some cleavage was determine the at positions −1 and −2 in the noncleaved strand a duplex that was a substrate for the enzyme or new cleavage the of reactions the was in the presence of the in Fig. 2 the topoisomerase I-mediated cleavage-ligation reaction with the unmodified partial duplex a acceptor afforded only a of ligation product and in good with a K.A. Hecht S.M. 1995; 34: PubMed Scopus Google Scholar). In incubation of topoisomerase I with partial duplexes containing a modified at position −1 or −2 good of ligation product 2 and in the presence of the The of acceptors with partial duplexes containing a at position −4 or −6 in the noncleaved strand afforded ligation product 2 The that the location of the modified bases within the noncleaved strand can be important of the site of cleavage. in which the efficiency and site of cleavage in the of acceptor the site of cleavage the position at which ligation I and Fig. the efficiency of cleavage was for of at positions −4 and −6 for at positions −1 and of of the noncleaved strand on the efficiency and site of DNA cleavage by topoisomerase of of cleavage at sites 1 and to the for the unmodified partial of cleavage at sites 1 and to the for the unmodified partial in a new the involving partial duplexes modified at sites on the noncleaved strand, we investigated the topoisomerase I-mediated cleavage reaction doubly modified partial The of reactions that been for the partial duplexes containing an unmodified scissile strand was with partial duplexes containing 3′-deoxyadenosine at position in Fig. at position +1 ligation of the of Fig. that this cleavage at site 1 and in an in cleavage at site 2 The of was with the The partial duplex having a single at the position +1 in the scissile strand and an unmodified noncleaved strand afforded enhanced ligation involving site 2 cleavage and The reactions involving partial duplexes containing both at position +1 in the scissile strand and positions −1 or −2 in the noncleaved strand increased site 2 cleavage as with the +1 on the scissile strand and In these the enzyme can catalyze the ligation reactions to and However, the at positions −4 or −6 in the noncleaved strand in to the +1 on the scissile strand in ligation at in the presence of the or acceptors and in the scissile strand were systematically substituted to determine which were important for and cleavage by topoisomerase In to the structural of a at position we were to topoisomerase I cleave the DNA backbone at the 2′ → 5′ in these in topoisomerase I can cleave and the 2′ → 5′ phosphodiester at the topoisomerase I high efficiency cleavage site in the modified partial The extent of ligation with this modified partial duplex was of that for the unmodified partial as by The partial duplexes containing a at position −1 in the scissile strand in to a at position −4 or −6 in the noncleaved strand were also substrates for the cleavage-ligation the enzyme the cleavage-ligation reaction to a extent to unmodified partial duplex and Fig. The partial duplex having a single at position −1 in the scissile strand was cleaved and with facility at site as by reactions in the presence of the acceptor at position −1 or −2 on the noncleaved strand with the at position −1 on the scissile strand further effect on the extent of cleavage and ligation at site 2 previously for the at position +1 of the acceptor was ligation product formed the substrate was modified at position −4 or −6 on the noncleaved strand in to the at position −1 on the scissile strand and of topoisomerase I-mediated cleavage-ligation of a substrate having a 2′ → 5′ at the cleavage The of ligation product with a substrate containing a single at position −1 in the scissile strand of the partial duplex was to that the unmodified partial duplex as a substrate, as by The partial duplex containing at position −1 in the scissile strand with a at position −4 or −6 in the noncleaved strand were also substrates for the the enzyme the cleavage-ligation reaction to a extent to unmodified partial oligonucleotides were to the effect on topoisomerase I-mediated cleavage and ligation of at position −2 of the scissile at position −2 not substrate cleavage and ligation by topoisomerase I an at position −1 on the noncleaved strand altered the cleavage site of the enzyme from site 1 to 2 in cleavage at site 2 as by a product in presence of the acceptor whereas all on the noncleaved strand afforded product with the in this at position −4 or −6 within the noncleaved strand enhanced the extent of cleavage and ligation at site 1 at position −4 of the scissile strand in ligation of the acceptor oligonucleotide by the substrate cleavage site Inclusion of 3′-deoxyadenosine at position −4 in the scissile strand in to at positions −1 or −2 in the noncleaved strand altered cleavage from site 1 to site 2 was a at −4 on the noncleaved strand, the product of cleavage and ligation at site 1 was with possible of the which could ligation by topoisomerase at −6 on the noncleaved strand ligation by the enzyme with both the and Although topoisomerase I is the substrate at site 2 in the presence of the position −6 on the noncleaved strand, this binding of the ligation to the normal The at position −6 in cleavage at site 1 and concomitant of product formation at positions −1 and −2 on the noncleaved strand that previously altered cleavage by topoisomerase I to site 2 as the (16Arslan T. Abraham A.T. Hecht S.M. Nucleosides Nucleotides. 1998; 17: 515-530Crossref PubMed Scopus (12) Google Scholar) or in with the presence of 3′-deoxyadenosine at position −4 on the scissile strand afforded cleavage at site 1 with at position −6 on the scissile strand on the noncleaved strand at positions −4 or −6 in cleavage at site 1 The substrate having on both at position −6 enhanced ligation at site 1 to the unmodified substrate In the presence of a the at position with at position −6 on the scissile strand, the of ligation due to partial cleavage at site 2. DNA topoisomerase I in the of DNA The enzyme can DNA by strand of the DNA substrate and and the cleaved strand via a covalent intermediate containing a (15Henningfeld K.A. Arslan T. Hecht S.M. J. Am. Chem. Soc. 1996; 118: 1701-1714Crossref Scopus (34) Google Scholar, K. Westergaard O. J. Biol. Chem. 1994; Full Text PDF PubMed Google Scholar). we reported that DNA topoisomerase I can the of DNA nucleotide and were in to of the DNA backbone (15Henningfeld K.A. Arslan T. Hecht S.M. J. Am. Chem. Soc. 1996; 118: 1701-1714Crossref Scopus (34) Google Scholar, K.A. Hecht S.M. 1995; 34: PubMed Scopus Google Scholar). The enzyme “suicide can ligation with DNA structural of the DNA S. J. Biol. Chem. Full Text PDF PubMed Google Scholar, DNA of the K. Scholar). The DNA duplex region for topoisomerase I-mediated reaction in was as an containing nucleotides on the scissile strand and nucleotides on the noncleaved strand (9Svejstrup J.Q. Christiansen K. Andersen A.H. Lund K. Westergaard O. J. Biol. Chem. 1990; 265: 12529-12535Abstract Full Text PDF PubMed Google Scholar) Fig. 1 is cleaved with the by eukaryotic topoisomerase I from DNA of the K. Scholar) and as a high efficiency site for DNA Bonven B.J. Westergaard O. PubMed Scopus Google Scholar). determine which nucleotides to the and facility of by the eukaryotic have utilized DNA containing nucleotides, abasic and Abasic sites at positions −2 to on the scissile strand rendered the substrate refractory to cleavage by topoisomerase abasic site at position −1 effect on enzyme (11Stevnsner T. Mortensen U.H. Westergaard O. Bonven B.J. J. Biol. Chem. 1989; 264: 10110-10113Abstract Full Text PDF PubMed Google Scholar). Recently, was reported that an abasic site on the noncleaved strand at positions −1 to −4 suppressed DNA cleavage at the normal site and new cleavage sites. abasic site at position −6 on the noncleaved strand increased the extent of cleavage (14Pourquier P. Ueng L.-M. Kohlhagen G. Mazumder A. Gupta M. Kohn K.W. Pommier Y. J. Biol. Chem. 1997; 272: 7792-7796Abstract Full Text Full Text PDF PubMed Scopus (194) Google Scholar). a substrate having two within the topoisomerase could a of the role of DNA on topoisomerase I-mediated and abasic sites be to the DNA duplex PubMed Scopus Google Scholar, T. M. J. Nucleosides Nucleotides. 1994; Scopus Google Scholar). In with the of abasic sites on DNA been reported that the of two within a duplex by only on the sites of J. Am. Chem. Soc. 114: Scopus Google Scholar, J. Am. Chem. Soc. 114: Scopus Google Scholar). In an we utilized as substrates a modified partial of which a single 3′-deoxyadenosine within the topoisomerase region on the scissile Depending on the location of the 3′-deoxyadenosine substitution, the facility of the topoisomerase I-mediated cleavage or ligation reaction was altered (16Arslan T. Abraham A.T. Hecht S.M. Nucleosides Nucleotides. 1998; 17: 515-530Crossref PubMed Scopus (12) Google Scholar). we have modified the partial duplex systematically to the of specific nucleotides by their 3′-deoxynucleotide Depending on the site of substitution, this can the of the enzyme by cleavage site or facility of cleavage or alterations in substrate binding by the were incorporated into the noncleaved strand to the effect on cleavage and These that at positions −1 and −2 alter the cleavage ligation was in presence of the acceptor but not in presence the The was in the of acceptor could be that these of the enzyme to cleavage site 1 the of the enzyme for this the in cleavage at site 2. of into positions −4 and −6 a effect on cleavage and ligation at site in the of ligation was for the substrate modified at positions −4 or was the that the extent of cleavage for these modified partial duplexes in the of acceptor oligonucleotides was only a of that for the unmodified partial that the acceptors can the process of cleavage and is that the formation of ligation from the substrates modified at positions −4 and −6 from cleavage at site of cleavage at site 2 be to be for position −4 on the noncleaved strand the to site 2 that position −2 to site 1 2 and were incorporated within the scissile with a at position were also systematically at positions and −6 within the scissile strand These modified oligonucleotides were utilized in coupled cleavage-ligation the modified scissile were with modified noncleaved to topoisomerase I function. at position +1 in the scissile strand altered substrate cleavage from site 1 to site to the of cleavage and abasic sites were incorporated at these positions (14Pourquier P. Ueng L.-M. Kohlhagen G. Mazumder A. Gupta M. Kohn K.W. Pommier Y. J. Biol. Chem. 1997; 272: 7792-7796Abstract Full Text Full Text PDF PubMed Scopus (194) Google Scholar). The were also to in which depurination of +1 of cleaved (11Stevnsner T. Mortensen U.H. Westergaard O. Bonven B.J. J. Biol. Chem. 1989; 264: 10110-10113Abstract Full Text PDF PubMed Google Scholar). In the presence of the topoisomerase I cleaved the partial duplex at site the intermediate ligation to form the at positions −1 or −2 effect on ligation the acceptor was whereas further at position −4 or −6 on the noncleaved strand ligation in the presence of the acceptor The of a 3′-deoxynucleotide at position −1 was of permitted investigation of the of the enzyme to cleave the DNA backbone at the site of an linkage. I effect cleavage at site 1 in this substrate of the cleavage was to site 2. The intermediate cleavage formed at sites 1 and 2 both ligation in the presence of the and acceptor for the substrates scissile strand of the partial duplexes at positions −1 and −2 on the noncleaved strand suppressed cleavage at site at position −4 and −6 suppressed cleavage at site 2. In the substrate having at position −2 cleavage and ligation at site 1 However, cleavage at site 2 was suppressed in with the of at position +1 on cleavage at site 1 and However, on the noncleaved strand alter the cleavage-ligation at sites 1 and 2. The substrate modified at position −1 on the noncleaved strand cleavage and ligation and only at site whereas at position −2 on the noncleaved strand also in cleavage and ligation but only at site at positions −4 or −6 on the noncleaved strand afforded substrates cleavage and ligation at site 1 was enhanced of 3′-deoxyadenosine at position −4 of the noncleaved strand in cleavage and ligation at site 2 is with the that the presence of the 3′-deoxynucleotide a effect on cleavage at the cleavage with the upon at position −2 on the scissile strand and at site −1 or −2 on the noncleaved strand further on the of cleavage-ligation at site 1 site 2. The in the presence of a at position −6 on the scissile Cleavage at site 1 in the presence of the substrate on the noncleaved The was also for partial duplexes having at position −4 or in the presence of at positions −1 or −2 on the noncleaved strand, which alter the cleavage from site 1 to site topoisomerase I cleaved the substrate at site of the substrates afforded a extent of cleavage and ligation the unmodified partial duplex substrate that of DNA at a site from the cleavage site can have a effect on DNA cleavage and was an analysis of the of complementary at the site on both strands. at positions −1 and −2 on both and the effect of topoisomerase I-mediated cleavage to sites and from the position of the were at sites from both cleavage the enhanced both the facility and of cleavage. was for at positions which cleavage-ligation to site 1 and for at positions which in of cleavage-ligation at site 1 at position −2 on the noncleaved strand cleavage to site 2 and ligation at this The the occurs a complementary and in the presence of a substrate modified at position −6 on the scissile at position −1 on the noncleaved strand cleavage to site 2 in The single involves substrates modified at position −6 on the scissile Because the of DNA at a specific site alter DNA the not to be the specific sites of topoisomerase I-DNA However, the of to DNA structural alterations that specific enzyme-DNA the and efficiency of DNA substrate cleavage and ligation by topoisomerase We for the of DNA topoisomerase I in these