The Role of the DNA Damage Response in Ataxia-Telangiectasia Syndrome

The DNA damage response (DDR) is a concerted response involving a myriad of pathways that cells elicit in the presence of DNA injuries. Patients bearing mutations in DDR genes have an increased cancer incidence derived from their diminished ability to respond to DNA damage, and the consequent increase in mutations. Intriguingly, mutations in ATM, the chief DDR regulator, can cause ataxia telangiectasia, a neurodegenerative disorder characterized by progressive loss of movement coordination, weak immune system, and increased cancer risk. The relationship between ATM and neural system development and degeneration remains to be fully elucidated and will be discussed in this chapter

Evolutionary Trace Annotation Server: automated enzyme function prediction in protein structures using 3D templates

Summary:The Evolutionary Trace Annotation (ETA) Server predicts enzymatic activity. ETA starts with a structure of unknown function, such as those from structural genomics, and with no prior knowledge of its mechanism uses the phylogenetic Evolutionary Trace (ET) method to extract key functional residues and propose a function-associated 3D motif, called a 3D template. ETA then searches previously annotated structures for geometric template matches that suggest molecular and thus functional mimicry. In order to maximize the predictive value of these matches, ETA next applies distinctive specificity filters—evolutionary similarity, function plurality and match reciprocity. In large scale controls on enzymes, prediction coverage is 43% but the positive predictive value rises to 92%, thus minimizing false annotations. Users may modify any search parameter, including the template. ETA thus expands the ET suite for protein structure annotation, and can contribute to the annotation efforts of metaservers

The AZFc region of the Y chromosome: at the crossroads between genetic diversity and male infertility

BACKGROUND: The three azoospermia factor (AZF) regions of the Y chromosome represent genomic niches for spermatogenesis genes. Yet, the most distal region, AZFc, is a major generator of large-scale variation in the human genome. Determining to what extent this variability affects spermatogenesis is a highly contentious topic in human reproduction. METHODS: In this review, an extensive characterization of the molecular mechanisms responsible for AZFc genotypical variation is undertaken. Such data are complemented with the assessment of the clinical consequences for male fertility imputable to the different AZFc variants. For this, a critical re-evaluation of 23 association studies was performed in order to extract unifying conclusions by curtailing methodological heterogeneities. RESULTS: Intrachromosomal homologous recombination mechanisms, either crossover or non-crossover based, are the main drivers for AZFc genetic diversity. In particular, rearrangements affecting gene dosage are the most likely to introduce phenotypical disruptions in the spermatogenic profile. In the specific cases of partial AZFc deletions, both the actual existence and the severity of the spermatogenic defect are dependent on the evolutionary background of the Y chromosome. CONCLUSIONS: AZFc is one of the most genetically dynamic regions in the human genome. This property may serve as counter against the genetic degeneracy associated with the lack of a meiotic partner. However, such strategy comes at a price: some rearrangements represent a risk factor or a de-facto causative agent of spermatogenic disruption. Interestingly, this precarious balance is modulated, among other yet unknown factors, by the evolutionary history of the Y chromosome

The Candida albicans Ku70 Modulates Telomere Length and Structure by Regulating Both Telomerase and Recombination

The heterodimeric Ku complex has been shown to participate in DNA repair and telomere regulation in a variety of organisms. Here we report a detailed characterization of the function of Ku70 in the diploid fungal pathogen Candida albicans. Both ku70 heterozygous and homozygous deletion mutants have a wild-type colony and cellular morphology, and are not sensitive to MMS or UV light. Interestingly, we observed complex effects of KU70 gene dosage on telomere lengths, with the KU70/ku70 heterozygotes exhibiting slightly shorter telomeres, and the ku70 null strain exhibiting long and heterogeneous telomeres. Analysis of combination mutants suggests that the telomere elongation in the ku70 null mutant is due mostly to unregulated telomerase action. In addition, elevated levels of extrachromosomal telomeric circles were detected in the null mutant, consistent with activation of aberrant telomeric recombination. Altogether, our observations point to multiple mechanisms of the Ku complex in telomerase regulation and telomere protection in C. albicans, and reveal interesting similarities and differences in the mechanisms of the Ku complex in disparate systems

Deletion of the Pichia pastoris KU70 Homologue Facilitates Platform Strain Generation for Gene Expression and Synthetic Biology

Targeted gene replacement to generate knock-outs and knock-ins is a commonly used method to study the function of unknown genes. In the methylotrophic yeast Pichia pastoris, the importance of specific gene targeting has increased since the genome sequencing projects of the most commonly used strains have been accomplished, but rapid progress in the field has been impeded by inefficient mechanisms for accurate integration. To improve gene targeting efficiency in P. pastoris, we identified and deleted the P. pastoris KU70 homologue. We observed a substantial increase in the targeting efficiency using the two commonly known and used integration loci HIS4 and ADE1, reaching over 90% targeting efficiencies with only 250-bp flanking homologous DNA. Although the ku70 deletion strain was noted to be more sensitive to UV rays than the corresponding wild-type strain, no lethality, severe growth retardation or loss of gene copy numbers could be detected during repetitive rounds of cultivation and induction of heterologous protein production. Furthermore, we demonstrated the use of the ku70 deletion strain for fast and simple screening of genes in the search of new auxotrophic markers by targeting dihydroxyacetone synthase and glycerol kinase genes. Precise knock-out strains for the well-known P. pastoris AOX1, ARG4 and HIS4 genes and a whole series of expression vectors were generated based on the wild-type platform strain, providing a broad spectrum of precise tools for both intracellular and secreted production of heterologous proteins utilizing various selection markers and integration strategies for targeted or random integration of single and multiple genes. The simplicity of targeted integration in the ku70 deletion strain will further support protein production strain generation and synthetic biology using P. pastoris strains as platform hosts

Mutant huntingtin impairs Ku70-mediated DNA repair

Mutant huntingtin prevents interaction of the DNA damage repair complex component Ku70 with damaged DNA, blocking repair of double-strand breaks

Accurate Protein Structure Annotation through Competitive Diffusion of Enzymatic Functions over a Network of Local Evolutionary Similarities

High-throughput Structural Genomics yields many new protein structures without known molecular function. This study aims to uncover these missing annotations by globally comparing select functional residues across the structural proteome. First, Evolutionary Trace Annotation, or ETA, identifies which proteins have local evolutionary and structural features in common; next, these proteins are linked together into a proteomic network of ETA similarities; then, starting from proteins with known functions, competing functional labels diffuse link-by-link over the entire network. Every node is thus assigned a likelihood z-score for every function, and the most significant one at each node wins and defines its annotation. In high-throughput controls, this competitive diffusion process recovered enzyme activity annotations with 99% and 97% accuracy at half-coverage for the third and fourth Enzyme Commission (EC) levels, respectively. This corresponds to false positive rates 4-fold lower than nearest-neighbor and 5-fold lower than sequence-based annotations. In practice, experimental validation of the predicted carboxylesterase activity in a protein from Staphylococcus aureus illustrated the effectiveness of this approach in the context of an increasingly drug-resistant microbe. This study further links molecular function to a small number of evolutionarily important residues recognizable by Evolutionary Tracing and it points to the specificity and sensitivity of functional annotation by competitive global network diffusion. A web server is at http://mammoth.bcm.tmc.edu/networks

Separation of Recombination and SOS Response in Escherichia coli RecA Suggests LexA Interaction Sites

RecA plays a key role in homologous recombination, the induction of the DNA damage response through LexA cleavage and the activity of error-prone polymerase in Escherichia coli. RecA interacts with multiple partners to achieve this pleiotropic role, but the structural location and sequence determinants involved in these multiple interactions remain mostly unknown. Here, in a first application to prokaryotes, Evolutionary Trace (ET) analysis identifies clusters of evolutionarily important surface amino acids involved in RecA functions. Some of these clusters match the known ATP binding, DNA binding, and RecA-RecA homo-dimerization sites, but others are novel. Mutation analysis at these sites disrupted either recombination or LexA cleavage. This highlights distinct functional sites specific for recombination and DNA damage response induction. Finally, our analysis reveals a composite site for LexA binding and cleavage, which is formed only on the active RecA filament. These new sites can provide new drug targets to modulate one or more RecA functions, with the potential to address the problem of evolution of antibiotic resistance at its root

Micromechanical Properties of Injection-Molded Starch–Wood Particle Composites

The micromechanical properties of injection molded starch–wood particle composites were investigated as a function of particle content and humidity conditions. The composite materials were characterized by scanning electron microscopy and X-ray diffraction methods. The microhardness of the composites was shown to increase notably with the concentration of the wood particles. In addition,creep behavior under the indenter and temperature dependence were evaluated in terms of the independent contribution of the starch matrix and the wood microparticles to the hardness value. The influence of drying time on the density and weight uptake of the injection-molded composites was highlighted. The results revealed the role of the mechanism of water evaporation, showing that the dependence of water uptake and temperature was greater for the starch–wood composites than for the pure starch sample. Experiments performed during the drying process at 70°C indicated that the wood in the starch composites did not prevent water loss from the samples.Peer reviewe