Abstract
The capacity of eukaryotic topoisomerase I to catalyze intra- and intermolecular DNA strand transfer via a two-step cleavage/ligation reaction was investigated by use of purified enzyme and defined DNA substrates. Topoisomerase I-mediated cleavage requires separate interaction with a duplex region encompassing the cleavage site (region A) and a duplex region located on the side holding the 5'-OH end generated by cleavage (region B). Cleaved topoisomerase I-DNA complexes containing enzyme covalently attached at internal and terminal positions were employed to characterize the intra- and intermolecular ligation reactions. Enzyme attached covalently at an internal position of a partially single-stranded DNA molecule is able to catalyze ligation of a complementary dinucleotide within region A in the absence of interaction with region B. Moreover, the dinucleotide confines the minimal DNA acceptor for intramolecular ligation. Topoisomerase I attached covalently to DNA at a terminal position can ligate the cleaved strand to heterologous duplex DNA regardless of sequence, whereas ligation does not proceed with single-stranded DNA. When these features are considered together with the observation that intermolecular ligation is inhibited by 1 M NaCl, it suggests that the reaction requires bipartite DNA interaction. A model is proposed that relates the bipartite DNA binding of eukaryotic topoisomerase I to the catalytic functions.
Highlights
Isomerase I-mediated cleavage requires separate inter- genes of !&trahymena (Bonven et al, 1985;Christiansen et al, action with a duplex region encompassing the cleavag1e987)and Dictiostylium (Nesset al., 1988).The cleavages occur site and a duplex region locatedon the side base in a hexadecameric sequence element present holding the6’-OHend generatedby cleavage. in several repeats withniunclease-hypersensitive sites.In vitro
To DNA at a terminal position can ligate the cleaved duplex requirements for DNA cleavage in the recognition sestrand to heterologousduplex DNA regardlessof se- quence were delimited by analyzing the interactionof the enquence, whereas ligation does not proceed with single-zyme with sets ofDNA substratesvarying successively by stranded DNA
When these features are considered to- single nucleotides at the 5‘- or 3’-end of either strand (Svejgether with the observation that intermolecular ligsattriuopnet al., 1990;Christiansen et al, 1993).Topoisomerase is inhibited by 1 M NaCl, it suggests that the reaction I-mediated cleavage was found to require enzyme interaction requires bipartiteDNA interaction.A model is proposed with two distinct DNA duplex regionslocated around thecleavthat relates the bipartiDteNA binding of eukaryotictopoisomerase I to the catalytic functions
Summary
Since double-stranded DNA acceptors are utilized without sequence specificity,while single-stranded topoisomerase I in 10 m~ Tris-HC1,pH 7.5, 5 m~ MgC12, and 5 rtl~ CaCl,, To exclude variation in the amount of cleaved donor topoisomerase I-DNA complexes in one set of ligation reactions, n cleavage reactions each containing 300 unita of topoisomerase I and 100 fmol of DNA substrate (enzyme:DNAmolar ratio of 6 1 ) were carried out in a DNA is not accepted, intermolecular ligation catalyzed by to- total volume of n x 50 pl.
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