Solving the riddle of codon usage preferences: a test for translational selection

Translational selection is responsible for the unequal usage of synonymous codons in protein coding genes in a wide variety of organisms. It is one of the most subtle and pervasive forces of molecular evolution, yet, establishing the underlying causes for its idiosyncratic behaviour across living kingdoms has proven elusive to researchers over the past 20 years. In this study, a statistical model for measuring translational selection in any given genome is developed, and the test is applied to 126 fully sequenced genomes, ranging from archaea to eukaryotes. It is shown that tRNA gene redundancy and genome size are interacting forces that ultimately determine the action of translational selection, and that an optimal genome size exists for which this kind of selection is maximal. Accordingly, genome size also presents upper and lower boundaries beyond which selection on codon usage is not possible. We propose a model where the coevolution of genome size and tRNA genes explains the observed patterns in translational selection in all living organisms. This model finally unifies our understanding of codon usage across prokaryotes and eukaryotes. Helicobacter pylori, Saccharomyces cerevisiae and Homo sapiens are codon usage paradigms that can be better understood under the proposed model

Unexpected correlations between gene expression and codon usage bias from microarray data for the whole Escherichia coli K-12 genome

Escherichia coli has long been regarded as a model organism in the study of codon usage bias (CUB). However, most studies in this organism regarding this topic have been computational or, when experimental, restricted to small datasets; particularly poor attention has been given to genes with low CUB. In this work, correspondence analysis on codon usage is used to classify E.coli genes into three groups, and the relationship between them and expression levels from microarray experiments is studied. These groups are: group 1, highly biased genes; group 2, moderately biased genes; and group 3, AT-rich genes with low CUB. It is shown that, surprisingly, there is a negative correlation between codon bias and expression levels for group 3 genes, i.e. genes with extremely low codon adaptation index (CAI) values are highly expressed, while group 2 show the lowest average expression levels and group 1 show the usual expected positive correlation between CAI and expression. This trend is maintained over all functional gene groups, seeming to contradict the E.coli–yeast paradigm on CUB. It is argued that these findings are still compatible with the mutation–selection balance hypothesis of codon usage and that E.coli genes form a dynamic system shaped by these factors

A structurally conserved motif in γ-herpesvirus uracil-DNA glycosylases elicits duplex nucleotide-flipping

Efficient γ-herpesvirus lytic phase replication requires a virally encoded UNG-type uracil-DNA glycosylase as a structural element of the viral replisome. Uniquely, γ-herpesvirus UNGs carry a seven or eight residue insertion of variable sequence in the otherwise highly conserved minor-groove DNA binding loop. In Epstein–Barr Virus [HHV-4] UNG, this motif forms a disc-shaped loop structure of unclear significance. To ascertain the biological role of the loop insertion, we determined the crystal structure of Kaposi’s sarcoma-associated herpesvirus [HHV-8] UNG (kUNG) in its product complex with a uracil-containing dsDNA, as well as two structures of kUNG in its apo state. We find the disc-like conformation is conserved, but only when the kUNG DNA-binding cleft is occupied. Surprisingly, kUNG uses this structure to flip the orphaned partner base of the substrate deoxyuridine out of the DNA duplex while retaining canonical UNG deoxyuridine-flipping and catalysis. The orphan base is stably posed in the DNA major groove which, due to DNA backbone manipulation by kUNG, is more open than in other UNG–dsDNA structures. Mutagenesis suggests a model in which the kUNG loop is pinned outside the DNA-binding cleft until DNA docking promotes rigid structuring of the loop and duplex nucleotide flipping, a novel observation for UNGs

Direct measurement of the substrate preference of uracil-DNA glycosylase

Site-directed mutants of the herpes simplex virus type 1 uracil-DNA glycosylase lacking catalytic activity have been used to probe the substrate recognition of this highly conserved and ubiquitous class of DNA-repair enzyme utilizing surface plasmon resonance. The residues aspartic acid-88 and histidine-210, implicated in the catalytic mechanism of the enzyme (Savva, R., McAuley-Hecht, K., Brown, T., and Pearl, L. (1995) Nature 373, 487–493; Slupphaug, G., Mol, C. D., Kavli, B., Arvai, A. S., Krokan, H. E. and Tainer, J. A. (1996) Nature 384, 87–92) were separately mutated to asparagine to allow investigations of substrate recognition in the absence of catalysis. The mutants were shown to be correctly folded and to lack catalytic activity. Binding to single- and double-stranded oligonucleotides, with or without uracil, was monitored by real-time biomolecular interaction analysis using surface plasmon resonance. Both mutants exhibited comparable rates of binding and dissociation on the same uracil-containing substrates. Interaction with single-stranded uracil-DNA was found to be stronger than with double-stranded uracil-DNA, and the binding to Gua:Ura mismatches was significantly stronger than that to Ade:Ura base pairs suggesting that the stability of the base pair determines the efficiency of interaction. Also, there was negligible interaction between the mutants and single- or double-stranded DNA lacking uracil, or with DNA containing abasic sites. These results suggest that it is uracil in the DNA, rather than DNA itself, that is recognized by the uracil-DNA glycosylases

Targeting uracil-DNA glycosylases for therapeutic outcomes using insights from virus evolution

Ung-type uracil-DNA glycosylases are frontline defenders of DNA sequence fidelity in bacteria, plants, and animals; Ungs also directly assist both innate and humoral immunity. Critically important in viral pathogenesis, whether acting for or against viral DNA persistence, Ungs also have therapeutic relevance to cancer, microbial, and parasitic diseases. Ung catalytic specificity is uniquely conserved, yet selective antiviral drugging of the Ung catalytic pocket is tractable. However, more promising precision therapy approaches present themselves via insights from viral strategies, including sequestration or adaptation of Ung for non-canonical roles. A universal Ung inhibition mechanism, converged upon by unrelated viruses, could also inform design of compounds to inhibit specific distinct Ungs. Extrapolating current developments, the character of such novel chemical entities is proposed

A comparative study of uracil-DNA glycosylases from human and herpes simplex virus type 1

Uracil-DNA glycosylase (UNG) is the key enzyme responsible for initiation of base excision repair. We have used both kinetic and binding assays for comparative analysis of UNG enzymes from humans and herpes simplex virus type 1 (HSV-1). Steady-state fluorescence assays showed that hUNG has a much higher specificity constant (kcat/Km) compared with the viral enzyme due to a lower Km. The binding of UNG to DNA was also studied using a catalytically inactive mutant of UNG and non-cleavable substrate analogs (2′-deoxypseudouridine and 2′-α-fluoro-2′-deoxyuridine). Equilibrium DNA binding revealed that both human and HSV-1 UNG enzymes bind to abasic DNA and both substrate analogs more weakly than to uracil-containing DNA. Structure determination of HSV-1 D88N/H210N UNG in complex with uracil revealed detailed information on substrate binding. Together, these results suggest that a significant proportion of the binding energy is provided by specific interactions with the target uracil. The kinetic parameters for human UNG indicate that it is likely to have activity against both U·A and U·G mismatches in vivo. Weak binding to abasic DNA also suggests that UNG activity is unlikely to be coupled to the subsequent common steps of base excision repair

Structural studies of a uracil-DNA glycosylase from herpes simplex virus type 1

The work presented in this thesis describes experiments carried out in order to determine the three-dimensional structure of a DNA repair enzyme, uracil-DNA glycosylase. An open reading frame, UL2, in the herpes simplex virus type 1 genome, is known to encode a uracil-DNA glycosylase. By sequence homology, there are three candidate start codons which might express a functional uracil-DNA glycosylase. Expression from two of these was attempted in Escherichia coli, using plasmids designed for high level production of recombinant proteins. The second candidate start codon produces high levels of a soluble, functional uracil-DNA glycosylase in Escherichia coli both in a native form, and as part of a fusion protein. Both the fusion and the native form of the enzyme have been purified to apparent homogeneity, as has a recombinantly expressed insoluble Escherichia coli uracil-DNA glycosylase. Preliminary attempts were made at deriving structural and functional information from the soluble, native recombinant herpes simplex enzyme with the use of circular dichroism. This form of uracil-DNA glycosylase has subsequently been crystallised in two ways, firstly as the free enzyme, and secondly in a complex with a single stranded DNA oligonucleotide. Extensive optimisation of the crystallisation parameters have been carried out in conjunction with modifications to the original purification protocol, and large, single crystals of both free, and DNA bound forms, suitable for X-ray diffraction studies are now readily reproduced. A systematic search for isomorphous heavy atom derivatives has been carried out for both types of crystal, and preliminary phases have been obtained for the DNA-bound form of the enzyme. This has enabled the calculation of an electron density map in which protein secondary structure features can be located. Improvement of this map will reveal the molecular structure of the enzyme/ DNA complex

The problem with Pyrimidines

The chemical properties of cytosine present cells with a serious informational ‘disease’, necessitating enzymes with exquisite specificity for deoxyuridine for its prevention and cure

DNA repair in three dimensions

Crystallographic studies of DNA repair enzymes are revealing a wide range of structural motifs used to recognize specific lesions in DNA, and are helping to unravel the catalytic mechanisms by which these lesions are repaired