Abstract
While the binding of adenyl-5'-yl imidodiphosphate (App(NH)p) to Drosophila melanogaster topoisomerase II induces a double-stranded DNA passage reaction, its nonhydrolyzable beta,gamma-imidodiphosphate bond prevents enzyme turnover (Osheroff, N., Shelton, E. R., and Brutlag, D. L. (1983) J. Biol. Chem. 258, 9536-9543). Therefore, this ATP analog was used to characterize the interactions between Drosophila topoisomerase II and DNA which occur after DNA strand passage but before enzyme turnover. In the presence of App(NH)p, a stable post-strand passage topoisomerase II-nucleic acid complex is formed when circular DNA substrates are employed. Although noncovalent in nature, these complexes are resistant to increases in ionic strength and show less than 5% dissociation under salt concentrations (greater than 500 mM) that disrupt 95% of the enzyme-DNA interactions formed in the absence of App(NH)p or under a variety of other conditions that do not support DNA strand passage. These results strongly suggest that the process of enzyme turnover not only regenerates the active conformation of topoisomerase II but also confers upon the enzyme the ability to disengage from its nucleic acid product. Experiments with linear DNA molecules indicate that after strand passage has taken place, topoisomerase II may be able to travel along its DNA substrate by a linear diffusion process that is independent of enzyme turnover. Further studies demonstrate that the regeneration of the enzyme's catalytic center does not require enzyme turnover, since topoisomerase II can cleave double-stranded DNA substrates after strand passage has taken place. Finally, while the 2'-OH and 3'-OH of ATP are important for its interaction with Drosophila topoisomerase II, neither are required for turnover.
Highlights
While the bindingof adenyl-5”yl imidodiphosphate stranded segmentof DNA through a transient break made in (App(NH)p) to Drosophila melanogaster topoisomer- a second helical segment [1,2,3]
In the presence of App(NH)p, a stablepost-strandpassage topoisomerase 11-nucleic acid complex is formed when circular DNA substrates areemployed. These complexes are resistant to increases in ionic strength and sholwess than 5%dissoaction of eukaryotic topoisomerase I1 appears to be a concerted reaction, it can be divided into several discrete steps: 1)binding of topoisomerase I1 to itsnucleic acid substrate; 2) double-stranded cleavage of the DNA, which is accompanied by the subsequent covalent attachment of the enzyme to the cleaved 5’ termini of the DNA [29, 30]; 3) double-stranded DNA passage through the break in thneucleic acid backbone, which is dependent on ATP binding to the enzyme [31, 32]; 4) religation of the cleaved DNA; and 5) enzyme turnover ( i e . the ability to reinitiate catalysis), which requires hydrolysis of the bound ATPcofactor [32,33,34]
Ciation under salt concentrations (>500 mM) that dis- I n vitro, thedouble-strandedDNA passage reaction of rupt 95%of the enzyme-DNA interactions formed in eukaryotic topoisomeraseTI has been studiedby a number of the absence of App(NH)p o r under a variety of other catalytic assays, including DNA relaxation [32], catenation conditions that do not support DNA strand passage. [33,34,35,36], decatenation [37], knotting [38], unknotting [31,39], These results strongly suggest that the processof en- and ATPhydrolysis [32, 37, 40]
Summary
Vol 261, No 21, Issue of July 25, pp. 9944-9950. 1986 Printed in U.S. A (Received for publication, August 5, 1985). Experiments with linear DNA molecules in- of the enzyme’s catalytic activity, the interactions between dicate that after strand passage has takpelnace, topo- Drosophilamelnnogaster topoisomerase I1 and DNA which isomerase I1 may be able to travel along iDtsNA sub- occur after strand passage but before turnover were characstrate by a linear diffusion process thaist independent terized This was accomplished by using an agarose gel elecoctswnfOhotfaoHirerntetahnanrrtDzunecedylqgrdrepnmueoa3aonisv’esrvoe-esdeOepratoarhuegHu.tnrieiblnoozalhfoynetavmA-oossefpTterrott.PathaiunsekFarodneruemeennordtezpvihmryleeaamrpscr,eeeosDs.’1ristnt1NFuac,cidnAneaniettaeialsttslflohuyyopedbt,riroietsciwsamtsrriohcaonemeitntrleeneesesrtatrqaeraftucarhstdtetieeiero2roeeI‘n1dts-hattAat3iirhsts6osTsia)psanPbphyusursoacte,tnurlreeawdieisnlcyqiioclsuglapoiincnwrsniodoijdhtutiteeicsncisafuchnoutbt-erpiiDsonptttndhNrhoauearAtctwtede-iaebisstfnihsntozdedycoAriimunapDbtgepiNlo(etnNA-uasHsrotnssrf)aatoptrnyova,,d’pene(oraad3dipss2nao)Dwos.msnTeNaehlghlrApyaeeadas,srresstoekualsIigry1unnezlefoatr(tsvbo3icleom2efr,. 2'-dATP, AdeArafTP (the 2"epimer of ATP), ADP, and CTP from TBE buffer During these studies, the level of topoisomerase I1 was P-L Biochemicals; ATP-yS from Boehringer Mannheim; bovine serum varied from 0.1 to 32 nM (0.05-16 units/reaction), while the concenalbumin (nuclease free) from Bethesda Research Laboratories; SDS tration of supercoiled pBR322 DNA was held constant a t 5 nM
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