title: A thymidine triphosphate shape analog lacking Watson-Crick pairing ability is replicated with high sequence selectivity.
abstract: Compound 1 (F), a nonpolar nucleoside analog that is isosteric with thymidine, has been proposed as a probe for the importance of hydrogen bonds in biological systems. Consistent with its lack of strong H-bond donors or acceptors, F is shown here by thermal denaturation studies to pair very poorly and with no significant selectivity among natural bases in DNA oligonucleotides. We report the synthesis of the 5'-triphosphate derivative of 1 and the study of its ability to be inserted into replicating DNA strands by the Klenow fragment (KF, exo- mutant) of Escherichia coli DNA polymerase I. We find that this nucleotide derivative (dFTP) is a surprisingly good substrate for KF; steady-state measurements indicate it is inserted into a template opposite adenine with efficiency (Vmax/Km) only 40-fold lower than dTTP. Moreover, it is inserted opposite A (relative to C, G, or T) with selectivity nearly as high as that observed for dTTP. Elongation of the strand past F in an F-A pair is associated with a brief pause, whereas that beyond A in the inverted A-F pair is not. Combined with data from studies with F in the template strand, the results show that KF can efficiently replicate a base pair (A-F/F-A) that is inherently very unstable, and the replication occurs with very high fidelity despite a lack of inherent base-pairing selectivity. The results suggest that hydrogen bonds may be less important in the fidelity of replication than commonly believed and that nucleotide/template shape complementarity may play a more important role than previously believed.
authors.affiliation: Department of Chemistry, University of Rochester, Rochester, NY 14627, USA.
authors.forename: S
authors.initials: S
authors.lastname: Moran
authors.forename: R X
authors.initials: RX
authors.lastname: Ren
authors.forename: E T
authors.initials: ET
authors.lastname: Kool
mesh_terms: D001482:Base Composition; D004256:DNA Polymerase I / Q000378:metabolism; D006860:Hydrogen Bonding; D007700:Kinetics; D009690:Nucleic Acid Conformation; D009691:Nucleic Acid Denaturation; D013379:Substrate Specificity; D013942:Thymine Nucleotides / Q000737:chemistry* / Q000378:metabolism
chemical_list: D013942:Thymine Nucleotides; D004256:DNA Polymerase I; C024157:thymidine 5'-triphosphate