THE PARADOXICAL INFLUENCE OF THYMINE ANALOGS ON RESTRICTION-ENDONUCLEASE CLEAVAGE OF OLIGODEOXYNUCLEOTIDES

Thymine residues in the DNA of eucaryotes may be replaced occasionally by uracil (U) or 5-(hydroxymethyl)uracil (H) as consequences of dUMP misincorporation or thymine oxidation, respectively. In this study, we constructed a series of 44-base oligonucleotides containing site-spec ific U or H residues and 5'-fluorescein labels in order to probe the i nfluence of such modifications on sequence-specific DNA-protein intera ctions using several type II restriction endonucleases. We find that s ubstitution within the recognition sites of several restriction endonu cleases increases initial cleavage velocity by up to an order of magni tude. These results contrast dramatically with several previous studie s which demonstrated that U substitution in short oligonucleotides inh ibits or prevents nuclease cleavage. We propose that this apparent par adox results because the rate-limiting step in the cleavage of longer oligonucleotides is product release whereas for shorter oligonucleotid es substrate binding is most probably rate-limiting. For longer oligon ucleotides and DNA, more rapid release of the cleaved, substituted oli gonucleotides results in more rapid turnover and a faster apparent cle avage rate. The sequence length at which the transition in rate-limiti ng step occurs likely corresponds to the size of the enzyme footprint on its DNA recognition site. We conclude that both U and H do perturb sequence-specific DNA-protein interactions, and the magnitude of this effect is site-dependent.