An NMR Investigation of the Effect of Hydrogen Bonding on the Rates of Rotation about the C-N Bonds in Urea and Thiourea

The interaction between urea and tetrabutylammonium acetate was investigated in dimethylformamide/ dimethyl sulfoxide solutions using ¹H and 15^N NMR. The chemical-shift behavior of the urea protons is consistent with a urea-acetate hydrogen-bonded complex involving both carboxylate oxygens and the urea hydrogens trans to the carbonyl oxygen with K_assoc = 120 ± 10. Line shape analysis of the temperature-dependent ¹H NMR spectra show that ∆G^‡ for rotation about the C-N bond of urea changes only slightly from 11.0 ± 0.1 to 11.2 ± 0.1 kcal/mol on 1:1 molar addition of tetrabutylammonium acetate to a dilute solution of urea. A parallel investigation of the interaction of thiourea with tetrabutylammonium acetate gave a binding constant, K_assoc = 90 ± 10. The ∆G^‡ for rotation about the C-N bond of thiourea was found to increase from 13.5 ± 0.1 to 14.0 ± 0.1 kcal/mol on 1:1 addition of tetrabutylammonium acetate to a dilute solution of thiourea in dimethylformamide/dimethyl sulfoxide. Measurements were also made of the self-association of several ureas and of ∆G^‡ for rotation about both C(O)-N bonds of 1,1-dimethylurea

Chromatin Assembly by DNA-translocating Motors

Chromatin assembly is required for the duplication of eukaryotic chromosomes and functions at the interface between cell-cycle progression and gene expression. The central machinery that mediates chromatin assembly consists of histone chaperones, which deliver histones to the DNA, and ATP-utilizing motor proteins, which are DNA-translocating factors that act in conjunction with the histone chaperones to mediate the deposition of histones into periodic nucleosome arrays. Here, we describe these factors and propose possible mechanisms by which DNA-translocating motors might catalyse chromatin assembly

Thermostability and excision activity of polymorphic forms of hOGG1

Abstract Objectives Reactive oxygen species (ROS) oxidize guanine residues in DNA to form 7,8-dihydro-oxo-2′-deoxyguanosine (8oxoG) lesions in the genome. Human 8-oxoguanine glycosylase-1 (hOGG1) recognizes and excises this highly mutagenic species when it is base-paired opposite a cytosine. We sought to characterize biochemically several hOGG1 variants that have been found in cancer tissues and cell lines, reasoning that if these variants have reduced repair capabilities, they could lead to an increased chance of mutagenesis and carcinogenesis. Results We have over-expressed and purified the R46Q, A85S, R154H, and S232T hOGG1 variants and have investigated their repair efficiency and thermostability. The hOGG1 variants showed only minor perturbations in the kinetics of 8oxoG excision relative to wild-type hOGG1. Thermal denaturation monitored by circular dichroism revealed that R46Q hOGG1 had a significantly lower Tm (36.6 °C) compared to the other hOGG1 variants (40.9 °C to 43.2 °C). Prolonged pre-incubation at 37 °C prior to the glycosylase assay dramatically reduces the excision activity of R46Q hOGG1, has a modest effect on wild-type hOGG1, and a negligible effect on A85S, R154H, and S232T hOGG1. The observed thermolability of hOGG1 variants was mostly alleviated by co-incubation with stoichiometric amounts of competitor DNA

Direct Visualization of a DNA Glycosylase Searching for Damage

DNA glycosylases preserve the integrity of genetic information by recognizing damaged bases in the genome and catalyzing their excision. It is unknown how DNA glycosylases locate covalently modified bases hidden in the DNA helix amongst vast numbers of normal bases. Here we employ atomic-force microscopy (AFM) with carbon nanotube probes to image search intermediates of human 8-oxoguanine DNA glycosylase (hOGG1) scanning DNA. We show that hOGG1 interrogates DNA at undamaged sites by inducing drastic kinks. The sharp DNA bending angle of these non-lesion-specific search intermediates closely matches that observed in the specific complex of 8-oxoguanine-containing DNA bound to hOGG1. These findings indicate that hOGG1 actively distorts DNA while searching for damaged bases

Identification of a New Uracil-DNA Glycosylase Family by Expression Cloning Using Synthetic Inhibitors

Background: The cellular environment exposes DNA to a wide variety of endogenous and exogenous reactive species that can damage DNA, thereby leading to genetic mutations. DNA glycosylases protect the integrity of the genome by catalyzing the first step in the base excision–repair of lesions in DNA. Results: Here, we report a strategy to conduct genome-wide screening for expressed DNA glycosylases, based on their ability to bind to a library of four synthetic inhibitors that target the enzyme\u27s active site. These inhibitors, used in conjunction with the in vitro expression cloning procedure, led to the identification of novel Xenopus and human proteins, xSMUG1 and hSMUG1, respectively, that efficiently excise uracil residues from DNA. Despite a lack of statistically significant overall sequence similarity to the two established classes of uracil-DNA glycosylases, the SMUG1 enzymes contain motifs that are hallmarks of a shared active-site structure and overall protein architecture. The unusual preference of SMUG1 for single-stranded rather than double-stranded DNA suggests a unique biological function in ridding the genome of uracil residues, which are potent endogenous mutagens. Conclusions: The ‘proteomics’ approach described here has led to the isolation of a new family of uracil-DNA glycosylases. The three classes of uracil-excising enzymes (SMUG1 being the most recently discovered) represent a striking example of structural and functional conservation in the almost complete absence of sequence conservation

Probing the Inverted Classroom: A Controlled Study of Teaching and Learning Outcomes in Undergraduate Engineering and Mathematics

The inverted or “flipped” classroom has begun to attract much attention among educators in an effort to combine the use of technology and traditional teaching techniques. One definition of the inverted classroom was provided by Lage, Platt, and Treglia1: “Inverting the classroom means that events that have traditionally taken place inside the classroom now take place outside the classroom and vice versa” (p.32). Bishop and Verleger2 provide an expanded view of the inverted classroom by defining it as “an educational technique that consists of two parts: interactive group learning activities inside the classroom, and direct computer-based individual instruction outside the classroom.

Excision of deaminated cytosine from the vertebrate genome: role of the SMUG1 uracil–DNA glycosylase

Gene-targeted mice deficient in the evolutionarily conserved uracil–DNA glycosylase encoded by the UNG gene surprisingly lack the mutator phenotype characteristic of bacterial and yeast ung(–) mutants. A complementary uracil–DNA glycosylase activity detected in ung(–/–) murine cells and tissues may be responsible for the repair of deaminated cytosine residues in vivo. Here, specific neutralizing antibodies were used to identify the SMUG1 enzyme as the major uracil–DNA glycosylase in UNG-deficient mice. SMUG1 is present at similar levels in cell nuclei of non-proliferating and proliferating tissues, indicating a replication- independent role in DNA repair. The SMUG1 enzyme is found in vertebrates and insects, whereas it is absent in nematodes, plants and fungi. We propose a model in which SMUG1 has evolved in higher eukaryotes as an anti-mutator distinct from the UNG enzyme, the latter being largely localized to replication foci in mammalian cells to counteract de novo dUMP incorporation into DNA

Structure and Specificity of the Vertebrate Anti-Mutator Uracil-DNA Glycosylase SMUG1

Cytosine deamination is a major promutagenic process, generating G:U mismatches that can cause transition mutations if not repaired. Uracil is also introduced into DNA via nonmutagenic incorporation of dUTP during replication. In bacteria, uracil is excised by uracil-DNA glycosylases (UDG) related to E. coli UNG, and UNG homologs are found in mammals and viruses. Ung knockout mice display no increase in mutation frequency due to a second UDG activity, SMUG1, which is specialized for antimutational uracil excision in mammalian cells. Remarkably, SMUG1 also excises the oxidation-damage product 5-hydroxymethyluracil (HmU), but like UNG is inactive against thymine (5-methyluracil), a chemical substructure of HmU. We have solved the crystal structure of SMUG1 complexed with DNA and base-excision products. This structure indicates a more invasive interaction with dsDNA than observed with other UDGs and reveals an elegant water displacement/replacement mechanism that allows SMUG1 to exclude thymine from its active site while accepting HmU