The Paradoxical Influence of Thymine Analogues on Restriction Endonuclease Cleavage of Oligodeoxynucleotides

Abstract

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-specific U or H residues and 5'-fluorescein labels in order to probe the influence of such modifications on sequence-specific DNA-protein interactions using several type II restriction endonucleases. We find that substitution within the recognition sites of several restriction endonucleases increases initial cleavage velocity by up to an order of magnitude. These results contrast dramatically with several previous studies which demonstrated that U substitution in short oligonucleotides inhibits or prevents nuclease cleavage. We propose that this apparent paradox results because the rate-limiting step in the cleavage of longer oligonucleotides is product release whereas for shorter oligonucleotides substrate binding is most probably rate-limiting. For longer oligonucleotides and DNA, more rapid release of the cleaved, substituted oligonucleotides results in more rapid turnover and a faster apparent cleavage rate. The sequence length at which the transition in rate-limiting 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.

Enzymatic Cleavage of Type II Restriction Endonucleases on the 2′-O-Methyl Nucleotide and Phosphorothioate Substituted DNA

“…In this study, the purchased synthetic phosphorothioate is a mixture of R p -form and S p -form diastereomers, and our results could be the fastest one among the R p -form cleavage and S p -form cleavage. We have observed that some substitutions increased cleavage efficiencies, such as XbaI-S7, SpeI-S2, SpeI-S3, SpeI-S5, SpeI-S6 and SpeI-S7, being consistent with previous reports [4] . The exact mechanism for such enhancement has not been given.…”

“…The majority of type II REs remains unexplored due primarily to the absence of structural information. To identify particular molecular interactions between REs and their duplex substrates and to obtain an understanding about the nuclease cleavage mechanisms, selective substitutions of nucleotide analogues for particular nucleotides of RE recognition sequences have been utilized extensively [4] , [5] , [6] , [7] .…”

Solid phase synthesis and restriction endonuclease cleavage of oligodeoxynucleotides containing 5-(hydroxymethyl)-cytosine

“…Oxidation of 5 m C to hm C could impact DNA-protein interactions because replacement of the hydrophobic methyl group by the hydrophilic hydroxymethyl group may prevent or alter the binding of proteins having hydrophobic binding clefts for the 5 m C methyl group. Recently, we demonstrated that replacement of T by hm U residues increases the dissociation rate of restriction nucleases from the cleaved substrates (29).…”

Nucleic Acid Ligands With Protein-like Side Chains: Modified Aptamers and Their Use as Diagnostic and Therapeutic Agents

“…72 Overall, the increase in stability to endonuclease degradation appears to be both larger and more consistent than that observed previously with other 5-position modifications. 86,87,88,89,90 …”