Relaxation of supercoiled phosphorothioate DNA by mammalian topoisomerases is inhibited in a base-specific manner.

M K Darby,H P Vosberg

doi:10.1016/s0021-9258(18)89292-4

Abstract

The nucleotide preferences of calf thymus topoisomerases I and II for recognition of supercoiled DNA have been assessed by the relaxation and cleavage of DNA containing base-specific phosphorothioate substitutions in one strand. The type I enzyme is inhibited to varying degrees by all modified DNAs, but most effectively (by approximately 60%) if deoxyguanosine 5'-O-(1-thiomonophosphate) (dGMP alpha S) is incorporated into negatively supercoiled DNA. A DNA in which all internucleotide linkages of one strand are phosphorothionate is relaxed, most probably via the unsubstituted strand. The type II enzyme is inhibited when deoxyadenosine 5'-O-(1-thiomonophosphate) (dAMP alpha S) or deoxyribosylthymine 5'-O-(1-thiomonophosphate) is incorporated into the DNA substrate, and the course of the relaxation reaction changes from a distributive mode to a predominantly processive mode. A fully substituted DNA is very poorly relaxed by the type II enzyme, illustrating the strict commitment of the enzyme to relaxation via double-strand cleavage. The sense of supercoiling does not affect the inhibition profile of either enzyme. DNA strand breaks introduced by type II topoisomerase in a normal control DNA or deoxycytidine 5'-O-(1-thiomonophosphate)-substituted DNA on treatment with sodium dodecyl sulfate at low ionic strength are prevented by pretreatment with 0.2 M NaCl. In contrast, breaks in DNA having either dAMP alpha S or all four phosphorothioate nucleotides incorporated in one strand are prevented only with higher NaCl concentrations. Thus indicating activity at the phosphorothioate linkage 5' to dA but not 5' to dC. We conclude that topoisomerase II activity occurs preferentially at sites possessing dAMP or dTMP, and that dGMP is involved in DNA recognition by topoisomerase I.

Highlights

Tively if deoxyguanosine 5‘0-1(-thiomonophosphate) is incorporated into negatively supercoiled DNA
Phosphorothioate DNA"A11 DNA molecules synthesized in vitro and used in these studies are hybrid duplexes, i.e. one strand is unsubstituted (the viral (+)-strand) and the other substituted according to the input deoxynucleotides during synthesis(the complementary (-)-strand)T. hus, fd(S)A DNA, for example, is a double-stranded circular fd DNA in which the (-)-strand is substitutewd ith a thiophosphodiester linkage 5' to every deoxyadenosine. (This DNA was previously referred to as fdAMPaS [16].) The DNA referred to here as M13(S)DNA is fully substituted inone strand, i.e. all internucleotide linkages are phosphorothioate
Studies on typIe1 restriction endonuclease activity towards phosphorothioate DNA have shown that activity is affected according tothe position of the modified linkagein the hexanucleotide recognition sequence [19]

Summary

RESULTS

Phosphorothioate DNA"A11 DNA molecules synthesized in vitro and used in these studies are hybrid duplexes, i.e. one strand is unsubstituted (the viral (+)-strand) and the other substituted according to the input deoxynucleotides during synthesis(the complementary (-)-strand)T. hus, fd(S)A DNA, for example, is a double-stranded circular fd DNA in which the (-)-strand is substitutewd ith a thiophosphodiester linkage 5' to every deoxyadenosine. (This DNA was previously referred to as fdAMPaS [16].) The DNA referred to here as M13(S)DNA is fully substituted inone strand, i.e. all internucleotide linkages are phosphorothioate. In this assay all phosphorothioate DNA was more slowly relaxed than unsubstituted DNA and the same relative order of activity was observed with these substrates. To the notion that the typeI enzyme can relax the DNA via Topoisomers of unique linking number of fd DNA, fd(S)A breakage and reclosure of the unsubstituted strand.Compa- DNA, and fd(S)CDNA, i.e. those substituted DNAs that are rable concentrations of thetype I1 enzymedisplayed no relaxed processively and distributively, respectively (Fig. 2 A ) , activity, suggesting that the thiodiesterlinkage was not bro- were prepared andincubated with calf thymus topoisomerase ken and that the enzyme was committed to a double strand 11. The reactionwas switch to a new binding site to effect relaxation when the essentially blocked by the substitution and under these assapyreferred (and more effective) sites were blocked

Cleavage of the Phosphorothioate Strandby Topoisomerase

ATP substitution on thecleavage of DNA compare withthe relaxation data?

DISCUSSION