CD spectra of wild-type (solid line; 2 µM) and -(2-hydroxyethyl)-cysteine-4 λ-repressor (dashed line; 2

<p><b>Copyright information:</b></p><p>Taken from "Switching DNA-binding specificity by unnatural amino acid substitution"</p><p>Nucleic Acids Research 2005;33(18):5896-5903.</p><p>Published online 13 Oct 2005</p><p>PMCID:PMC1258173.</p><p>© The Author 2005. Published by Oxford University Press. All rights reserved</p>4 µM). Experimental details are in Materials and Methods

Fluorescence emission spectra of wild-type (solid line; 2 µM) and -(2-hydroxyethyl)-cysteine-4 λ-repressor (dashed line; 2

<p><b>Copyright information:</b></p><p>Taken from "Switching DNA-binding specificity by unnatural amino acid substitution"</p><p>Nucleic Acids Research 2005;33(18):5896-5903.</p><p>Published online 13 Oct 2005</p><p>PMCID:PMC1258173.</p><p>© The Author 2005. Published by Oxford University Press. All rights reserved</p>4 µM). Experimental details are in Materials and Methods

Crystal structure of λ-repressor N-terminal domain crystal structure complexed with O1

<p><b>Copyright information:</b></p><p>Taken from "Switching DNA-binding specificity by unnatural amino acid substitution"</p><p>Nucleic Acids Research 2005;33(18):5896-5903.</p><p>Published online 13 Oct 2005</p><p>PMCID:PMC1258173.</p><p>© The Author 2005. Published by Oxford University Press. All rights reserved</p> Lysine-4 is shown in green and asparagine-55 is shown in cyan. The base pair CG6 is shown in space filling representation. Lower panel depicts the sequence of O1. The top half is the consensus half. Of the two strands, the top of left strand is the 5′ end

A Synthetic Peptide Mimic of λ-Cro shows Sequence-Specific Binding <i>in Vitro</i> and <i>in Vivo</i>

Development of small synthetic transcription factors is important for future cellular engineering and therapeutics. This article describes the chemical synthesis of α-amino-isobutyric acid (Aib) substituted, conformationally constrained, helical peptide mimics of <i>Cro</i> protein from bacteriophage λ that encompasses the DNA recognition elements. The Aib substituted constrained helical peptide monomer shows a moderately reduced dissociation constant compared to the corresponding unsubstituted wild type peptide. A suitably cross-linked dimeric version of the peptide, mimicking the dimeric protein, recapitulates some of the important features of <i>Cro</i>. It binds to the operator site O_R3, a high affinity <i>Cro</i> binding site in the λ genome, with good affinity and single base-pair discrimination specificity. A dimeric version of an even shorter peptide mimic spanning only the recognition helix of the helix-turn-helix motif of the <i>Cro</i> protein was created following the same design principles. This dimeric peptide binds to O_R3 with affinity greater than that of the longer version. Chemical shift perturbation experiments show that the binding mode of this peptide dimer to the cognate operator site sequence is similar to the wild type <i>Cro</i> protein. A Green Fluorescent Protein based reporter assay <i>in vivo</i> reveals that the peptide dimer binds the operator site sequences with considerable selectivity and inhibits gene expression. Peptide mimics designed in this way may provide a future framework for creating effective synthetic transcription factors

Divergent Mechanisms for Enzymatic Excision of 5‑Formylcytosine and 5‑Carboxylcytosine from DNA

5-Methyl­cytosine (mC) is an epigenetic mark that impacts transcription, development, and genome stability, and aberrant DNA methylation contributes to aging and cancer. Active DNA demethylation involves stepwise oxidation of mC to 5-hydroxy­methyl­cytosine, 5-formyl­cytosine (fC), and potentially 5-carboxyl­cytosine (caC), excision of fC or caC by thymine DNA glycosylase (TDG), and restoration of cytosine via follow-on base excision repair. Here, we investigate the mechanism for TDG excision of fC and caC. We find that 5-carboxyl-2′-deoxy­cytidine ionizes with p<i>K</i> _a values of 4.28 (N3) and 2.45 (carboxyl), confirming that caC exists as a mono­anion at physiological pH. Calculations do not support the proposal that G·fC and G·caC base pairs adopt a wobble structure that is recognized by TDG. Previous studies show that <i>N</i>-glycosidic bond hydrolysis follows a stepwise (S_N1) mechanism, and that TDG activity increases with pyrimidine N1 acidity, that is, leaving group quality of the target base. Calculations here show that fC and the neutral tautomers of caC are acidic relative to other TDG substrates, but the caC monoanion exhibits poor acidity and likely resists TDG excision. While fC activity is independent of pH, caC excision is acid-catalyzed, and the pH profile indicates that caC ionizes in the enzyme–substrate complex with an apparent p<i>K</i> _a of 5.8, likely at N3. Mutational analysis reveals that Asn191 is essential for excision of caC but dispensable for fC activity, indicating that N191 may stabilize N3-protonated forms of caC to facilitate acid catalysis and suggesting that N191A-TDG could potentially be useful for studying DNA demethylation in cells

Nanoscale Characterization of Interaction of APOBEC3G with RNA

The human cytidine deaminase APOBEC3G (A3G) is a potent inhibitor of the HIV-1 virus in the absence of viral infectivity factor (Vif). The molecular mechanism of A3G antiviral activity is primarily attributed to deamination of single-stranded DNA (ssDNA); however, the nondeamination mechanism also contributes to HIV-1 restriction. The interaction of A3G with ssDNA and RNA is required for its antiviral activity. Here we used atomic force microscopy to directly visualize A3G–RNA and A3G–ssDNA complexes and compare them to each other. Our results showed that A3G in A3G–RNA complexes exists primarily in monomeric–dimeric states, similar to its stoichiometry in complexes with ssDNA. New A3G–RNA complexes in which A3G binds to two RNA molecules were identified. These data suggest the existence of two separate RNA binding sites on A3G. Such complexes were not observed with ssDNA substrates. Time-lapse high-speed atomic force microscopy was applied to characterize the dynamics of the complexes. The data revealed that the two RNA binding sites have different affinities for A3G. On the basis of the obtained results, a model for the interaction of A3G with RNA is proposed