Impact of sugar pucker on base pair and mispair stability.

The selection of nucleoside triphosphates by a polymerase is controlled by several energetic and structural features, including base pairing geometry as well as sugar structure and conformation. Whereas base pairing has been considered exhaustively, substantially less is known about the role of sugar modifications for both nucleotide incorporation and primer extension. In this study, we synthesized oligonucleotides containing 2'-fluoro-modified nucleosides with constrained sugar pucker in an internucleotide position and, for the first time, at a primer 3'-end. The thermodynamic stability of these duplexes was examined. The nucleoside 2'-deoxy-2'-fluoroarabinofuranosyluracil [U(2'F(ara))] favors the 2'-endo conformation (DNA-like), while 2'-deoxy-2'-fluororibofuranosyluracil [U(2'F(ribo))] favors the 3'-endo conformation (RNA-like). Oligonucleotides containing U(2'F(ara)) have slightly higher melting temperatures (T(m)) than those containing U(2'F(ribo)) when located in internucleotide positions or at the 3'-end and when correctly paired with adenine or mispaired with guanine. However, both modifications decrease the magnitude of DeltaH degrees and DeltaS degrees for duplex formation in all sequence contexts. In examining the thermodynamic properties for this set of oligonucleotides, we find entropy-enthalpy compensation is apparent. Our thermodynamic findings led to a series of experiments with DNA ligase that reveal, contrary to expectation based upon observed T(m) values, that the duplex containing the U(2'F(ribo)) analogue is more easily ligated. The 2'-fluoro-2'-deoxynucleosides examined here are valuable probes of the impact of sugar constraint and are also members of an important class of antitumor and antiviral agents. The data reported here may facilitate an understanding of the biological properties of these agents, as well as the contribution of sugar conformation to replication fidelity.

Cloning and characterization of Rhodotorula glutinis thymine hydroxylase.

Thymine hydroxylase (TH) is a member of the alpha-ketoglutarate-dependent nonheme iron dioxygenase family that includes a series of DNA repair proteins including alkB. Substantial interest in this family of enzymes derives from their capacity to modify DNA bases and precursors by oxidation. Previously, a sequence has been published for cloned Rhodotorula glutinis TH. However, the minimal reported activity of this enzyme, coupled with inconsistencies with previously published mass spectrometry data, compelled us to reexamine TH. The sequence reported here differs from the previously reported sequence at two amino acid positions and is consistent with previously reported mass spectrometry data. The cloned enzyme characterized in this report displayed substantial activity, indicating that the sequence differences are critical for activity. The substrate selectivity of TH against a series of pyrimidine analogues is consistent with that reported for the wild-type enzyme and, in part, explains the mode of selection of uracil analogues. A preliminary model of the active site has been constructed for the purposes of comparing TH with other members of this family. TH and alkB share in common the capacity to oxidize N-methyl groups. However, TH has the added capacity to oxidize the 5-methyl group of thymine, a property that is potentially important for enzymes that could act on DNA and modify DNA-protein interactions.