A Multimodal Approach towards Genomic Identification of Protein Inhibitors of Uracil-DNA Glycosylase.

DNA-mimicking proteins encoded by viruses can modulate processes such as innate cellular immunity. An example is Ung-family uracil-DNA glycosylase inhibition, which prevents Ung-mediated degradation via the stoichiometric protein blockade of the Ung DNA-binding cleft. This is significant where uracil-DNA is a key determinant in the replication and distribution of virus genomes.

Cryo-EM Structures of Two Bacteriophage Portal Proteins Provide Insights for Antimicrobial Phage Engineering.

Widespread antibiotic resistance has returned attention to bacteriophages as a means of managing bacterial pathogenesis. Synthetic biology approaches to engineer phages have demonstrated genomic editing to broaden natural host ranges, or to optimise microbicidal action. Gram positive pathogens cause serious pastoral animal and human infections that are especially lethal in newborns.

The Essential Co-Option of Uracil-DNA Glycosylases by Herpesviruses Invites Novel Antiviral Design.

Vast evolutionary distances separate the known herpesviruses, adapted to colonise specialised cells in predominantly vertebrate hosts. Nevertheless, the distinct herpesvirus families share recognisably related genomic attributes. The taxonomic Family includes many important human and animal pathogens.

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.

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.

Architecturally diverse proteins converge on an analogous mechanism to inactivate Uracil-DNA glycosylase.

Uracil-DNA glycosylase (UDG) compromises the replication strategies of diverse viruses from unrelated lineages. Virally encoded proteins therefore exist to limit, inhibit or target UDG activity for proteolysis. Viral proteins targeting UDG, such as the bacteriophage proteins ugi, and p56, and the HIV-1 protein Vpr, share no sequence similarity, and are not structurally homologous.

Engineering human MEK-1 for structural studies: A case study of combinatorial domain hunting.

Structural biology studies typically require large quantities of pure, soluble protein. Currently the most widely-used method for obtaining such protein involves the use of bioinformatics and experimental methods to design constructs of the target, which are cloned and expressed. Recently an alternative approach has emerged, which involves random fragmentation of the gene of interest and screening for well-expressing fragments.

A structural and functional dissection of the cardiac stress response factor MS1.

MS1 is a protein predominantly expressed in cardiac and skeletal muscle that is upregulated in response to stress and contributes to development of hypertrophy. In the aortic banding model of left ventricular hypertrophy, its cardiac expression was significantly upregulated within 1 h. Its function is postulated to depend on its F-actin binding ability, located to the C-terminal half of the protein, which promotes stabilization of F-actin in the cell thus releasing myocardin-related transcription factors to the nucleus where they stimulate transcription in cooperation with serum response factor.

A combinatorial method to enable detailed investigation of protein-protein interactions.

Background: Successful structural investigations of protein-protein interactions can be facilitated by studying only the core interacting regions of the constituent proteins. However, attempting the discovery of stable core complexes using informed trial-and-error approaches can prove time and resource intensive.

Methods: We describe a valuable extension of combinatorial domain hunting (CDH), a technology for the timely elucidation of soluble protein truncations.

Crystal structure of a KSHV-SOX-DNA complex: insights into the molecular mechanisms underlying DNase activity and host shutoff.

The early lytic phase of Kaposi's sarcoma herpesvirus infection is characterized by viral replication and the global degradation (shutoff) of host mRNA. Key to both activities is the virally encoded alkaline exonuclease KSHV SOX. While the DNase activity of KSHV SOX is required for the resolution of viral genomic DNA as a precursor to encapsidation, its exact involvement in host shutoff remains to be determined.

DNA fragmentation based combinatorial approaches to soluble protein expression Part II: library expression, screening and scale-up.

In this second of a two-part review encompassing random, combinatorial methods for soluble protein 'domain hunting', we focus upon the expression screening from DNA fragment libraries. Given a library of domain length-encoding DNA fragments assembled in expression vectors, it is necessary to devise reliable means to screen the sample DNA fragment population to find those that express stable, soluble target protein fragments, suitable for the required downstream aims. This review summarizes a variety of alternative strategies that have been employed to identify such stable truncates of full-length proteins.

DNA fragmentation-based combinatorial approaches to soluble protein expression Part I. Generating DNA fragment libraries.

In addressing a new drug discovery target, the generation of tractable protein substrates for functional and structural analyses can represent a significant hurdle. Traditional approaches rely on protein expression trials of multiple variants in various systems, frequently with limited success. The increasing knowledge base derived from genomics and structural proteomics initiatives assists the bioinformatics-led design of these experiments.

Combinatorial Domain Hunting: An effective approach for the identification of soluble protein domains adaptable to high-throughput applications.

Exploitation of potential new targets for drug and vaccine development has an absolute requirement for multimilligram quantities of soluble protein. While recombinant expression of full-length proteins is frequently problematic, high-yield soluble expression of functional subconstructs is an effective alternative, so long as appropriate termini can be identified. Bioinformatics localizes domains, but doesn't predict boundaries with sufficient accuracy, so that subconstructs are typically found by trial and error.

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 (k(cat)/K(m)) compared with the viral enzyme due to a lower K(m).

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.

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.

Crystal structure of the Escherichia coli dcm very-short-patch DNA repair endonuclease bound to its reaction product-site in a DNA superhelix.

Very-short-patch repair (Vsr) enzymes occur in a variety of bacteria, where they initiate nucleotide excision repair of G:T mismatches arising by deamination of 5-methyl-cytosines in specific regulatory sequences. We have now determined the structure of the archetypal dcm-Vsr endonuclease from Escherichia coli bound to the cleaved authentic hemi-deaminated/hemi-methylated dcm sequence 5'-C-OH-3' 5'-p-T-p-A-p-G-p-G-3'/3'-G-p-G-p-T-p(Me5)C-p-C formed by self-assembly of a 12mer oligonucleotide into a continuous nicked DNA superhelix. The structure reveals the presence of a Hoogsteen base pair within the deaminated recognition sequence and the substantial distortions of the DNA that accompany Vsr binding to product sites.

The mechanism of DNA repair by uracil-DNA glycosylase: studies using nucleotide analogues.

2',4'-Dideoxy-4'-methyleneuridine incorporated into oligodeoxynucleotides forms regular B-DNA duplexes as shown by Tm and CD measurements. Such oligomers are not cleaved by the DNA repair enzyme, UDG, which cleaves the glycosylic bond in dU but not in dT nor in dC nucleosides in single stranded and double stranded DNA. Differential binding of oligomers containing carbadU, 4'-thiodU, and dU residues to wild type and mutant UDG proteins identify an essential role for the furanose 4'-oxygen in recognition and cleavage of dU residues in DNA.

Crystal structure of a thwarted mismatch glycosylase DNA repair complex.

The bacterial mismatch-specific uracil-DNA glycosylase (MUG) and eukaryotic thymine-DNA glycosylase (TDG) enzymes form a homologous family of DNA glycosylases that initiate base-excision repair of G:U/T mismatches. Despite low sequence homology, the MUG/TDG enzymes are structurally related to the uracil-DNA glycosylase enzymes, but have a very different mechanism for substrate recognition. We have now determined the crystal structure of the Escherichia coli MUG enzyme complexed with an oligonucleotide containing a non-hydrolysable deoxyuridine analogue mismatched with guanine, providing the first structure of an intact substrate-nucleotide productively bound to a hydrolytic DNA glycosylase.

Structure of a DNA base-excision product resembling a cisplatin inter-strand adduct.

Base-excision of a self-complementary oligonucleotide with central G:T mismatches by the G:T/U-specific mismatch DNA glycosylase (MUG), generates an unusual DNA structure which is remarkably similar in conformation to an interstrand DNA adduct of the anti-tumor drug cis-diamminedichloroplatinum. The abasic sugars generated by excision of the mismatched thymines are extruded from the double-helix, and the 'widowed' deoxyguanosines rotate so that their N7 and O6 groups protrude into the minor groove of the duplex and restack in an interleaved intercalative geometry, generating a kink in the helix axis.

Crystal structure of a G:T/U mismatch-specific DNA glycosylase: mismatch recognition by complementary-strand interactions.

G:U mismatches resulting from deamination of cytosine are the most common promutagenic lesions occurring in DNA. Uracil is removed in a base-excision repair pathway by uracil DNA-glycosylase (UDG), which excises uracil from both single- and double-stranded DNA. Recently, a biochemically distinct family of DNA repair enzymes has been identified, which excises both uracil and thymine, but only from mispairs with guanine.

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.

Uracil-DNA glycosylase activities in hyperthermophilic micro-organisms.

Hyperthermophiles exist in conditions which present an increased threat to the informational integrity of their DNA, particularly by hydrolytic damage. As in mesophilic organisms, specific activities must exist to restore and protect this template function of DNA. In this study we have demonstrated the presence of thermally stable uracil-DNA glycosylase activities in seven hyperthermophiles; one bacterial: Thermotoga maritima, and six archaeal: Sulfolobus solfataricus, Sulfolobus shibatae, Sulfolobus acidocaldarius, Thermococcus litoralis, Pyrococcus furiosus and Pyrobaculum islandicum.