The Steric Hypothesis for DNA Replication and Fluorine Hydrogen Bonding Revisited in Light of Structural Data

Abstract

In DNA, bases pair in a molecular interaction that is both highly predictable and exquisitely specific. Therefore researchers have generally believed that the insertion of the matching nucleotide opposite a template base by DNA polymerases (pols) required Watson-Crick (W-C) hydrogen bond formation. However pioneering work by Kool and co-workers using hydrophobic base analogs such as the thymine (T) isostere 2,4-difluorotoluene (F) showed that shape rather than H-bonding served as the primary source of specificity in DNA replication by certain pols. This steric hypothesis for DNA replication has gained popularity, perhaps discouraging further experimental studies to address potential limitations of this new idea. The idea that shape trumps H-bonding in terms of pol selectivity largely hinges on the belief that fluorine is a poor H-bond acceptor. However, the shape complementarity model was embraced in the absence of any detailed structural data for match (F:A) and mismatch pairs (F:G, F:C, F:T) in DNA duplexes or at active sites of pols. Although the F and T nucleosides are roughly isosteric, it is unclear whether F:A and T:A pairs exhibit similar geometries. If the F:A pair is devoid of H-bonding, it will be notably wider than a T:A pair. Because shape and size and H-bonding are intimately related, it may not be possible to separate these two properties. Thus the geometries of an isolated F:A pair in water may differ considerably from an F:A pair embedded in a stretch of duplex DNA, at the tight active site of an A-family replicative pol, or within the spacious active site of a Y-family translesion pol. The shape complementarity model may have more significance for pol accuracy than efficiency: this model appears to be most relevant for replicative pols that use specific residues to probe the identity of the nascent base pair from the minor groove side. However, researchers have not fully considered the importance of such interactions that include H-bonds compared with W-C H-bonds in terms of pol fidelity and the shape complementarity model. This Account revisits the steric hypothesis for DNA replication in light of recent structural data and discusses the role of fluorine as an H-bond acceptor. Over the last 5 years, crystal structures have emerged for nucleic acid duplexes with F paired opposite to natural bases or located at the active sites of DNA pols. These data permit a more nuanced understanding of the role of shape in DNA replication and the capacity of fluorine to form H-bonds. These studies and additional research involving RNA or other fluorine-containing nucleoside analogs within duplexes indicate that fluorine engages in H-bonding in many cases. Although T and F are isosteric at the nucleoside level, replacement of a natural base by F in pairs often changes their shapes and sizes, and dF in DNA behaves differently from rF in RNA. Similarly, the pairing geometries observed for F and T opposite dATP, dGTP, dTTP, or dCTP and their H-bonding patterns at the active site of a replicative pol differ considerably.