While the roles of 5-methyl-cytosine and 5-hydroxymethyl-cytosine in epigenetic regulation of gene expression are well-established, the functional effects of 5-formyl-cytosine and 5-carboxyl-cytosine in the genome on transcription are not clear. Here we report the first systematic study of the effects of five different forms of cytosine in DNA on mammalian and yeast RNA polymerase II transcription, providing new insights into potential functional interplay between cytosine methylation status and transcription.

Single turnover studies on HIV reverse transcriptase suggest that nucleoside analogs bind more tightly to the enzyme than normal substrates, contrary to rational structural predictions. Here we resolve these controversies by monitoring the kinetics of nucleotideinduced changes in enzyme structure. We show that the specificity constant for incorporation of a normal nucleotide (dCTP) is determined solely by the rate of binding (including isomerization) because isomerization to the closed complex commits the substrate to react. In contrast, a nucleoside analog (3TC-TP, triphosphate form of lamivudine) is incorporated slowly, allowing the conformational change to come to equilibrium and revealing tight nucleotide binding. Our data reconcile previously conflicting reports suggesting that nucleotide analogs bind tighter than normal nucleotides. Rather, dCTP and 3TC-TP bind with nearly equal affinities, but the binding of dCTP never reaches equilibrium. Discrimination against 3TC-TP is based on the slower rate of incorporation due to misalignment of the substrate and/or catalytic residues.human immunodeficiency virus | pre-steady-state kinetics | substrate specificity | nucleoside analogs | polymerase fidelity T he contribution of substrate-induced structure changes toward enzyme specificity and efficiency has long been debated (1). Although considerable attention has been given to the small conformational changes that are thought to effect catalysis from the closed enzyme-substrate complex (2, 3), little is known about the role of the larger changes in structure from an open to a closed complex following substrate binding. Like most enzymes, structural analysis of HIV reverse transcriptase (RT) reveals a large conformational change after nucleotide binding (4, 5). However, the role of substrate-induced conformational changes in specificity is controversial.Nucleotide binding and incorporation by HIV RT was initially characterized by Kati et al. (6) using rapid chemical-quench-flow methods. Examination of the nucleotide concentration dependence of the rate of polymerization under single turnover conditions provided an apparent nucleotide dissociation constant (K d;app ) and a maximum rate of nucleotide incorporation (k pol ) according to Scheme 1.Interpretation of these data depended upon the simplifying assumption that polymerization was governed by a single ratelimiting step (k pol ), that nucleotide binding occurred as a rapid equilibrium (K d;app ), and that reactions following polymerization (pyrophosphate release and translocation) were fast. These parameters provide the best measurements to define k cat and K m values that apply to processive DNA synthesis where the polymerase incorporates nucleotides sequentially to extend a growing polymer. This model and method of analysis have since been adopted throughout the polymerase field to assign values for nucleotide binding (K d;app ) and incorporation (k pol ) governing specificity, where k cat ∕K m ¼ k pol ∕K d;app . Although the kinetics of inhibition of HIV RT ...

Maintaining high transcriptional fidelity is essential to life. For all eukaryotic organisms, RNA polymerase II (Pol II) is responsible for messenger RNA synthesis from the DNA template. Three key checkpoint steps are important in controlling Pol II transcriptional fidelity: nucleotide selection and incorporation, RNA transcript extension, and proofreading. Some types of DNA damage significantly reduce transcriptional fidelity. However, the chemical interactions governing each individual checkpoint step of Pol II transcriptional fidelity and the molecular basis of how subtle DNA base damage leads to significant losses of transcriptional fidelity are not fully understood. Here we use a series of “hydrogen bond deficient” nucleoside analogs to dissect chemical interactions governing Pol II transcriptional fidelity. We find that whereas hydrogen bonds between a Watson-Crick base pair of template DNA and incoming NTP are critical for efficient incorporation, they are not required for efficient transcript extension from this matched 3’-RNA end. In sharp contrast, the fidelity of extension is strongly dependent on the discrimination of an incorrect pattern of hydrogen bonds. We show that U:T wobble base interactions are critical to prevent extension of this mismatch by Pol II. Additionally, both hydrogen bonding and base stacking play important roles in controlling Pol II proofreading activity. Strong base stacking at the 3’-RNA terminus can compensate for loss of hydrogen bonds. Finally, we show that Pol II can distinguish very subtle size differences in template bases. The current work provides the first systematic evaluation of electrostatic and steric effects in controlling Pol II transcriptional fidelity.