The Zn-Finger of Saccharomyces cerevisiae Rad18 and Its Adjacent Region Mediate Interaction with Rad5

DNA damages that hinder the movement of the replication complex can ultimately lead to cell death. To avoid that, cells possess several DNA damage bypass mechanisms. The Rad18 ubiquitin ligase controls error-free and mutagenic pathways that help the replication complex to bypass DNA lesions by monoubiquitylating PCNA at stalled replication forks. In Saccharomyces cerevisiae, two of the Rad18 governed pathways are activated by monoubiquitylated PCNA and they involve translesion synthesis polymerases, whereas a third pathway needs subsequent polyubiquitylation of the same PCNA residue by another ubiquitin ligase the Rad5 protein, and it employs template switching. The goal of this study was to dissect the regulatory role of the multidomain Rad18 in DNA damage bypass using a structure-function based approach. Investigating deletion and point mutant RAD18 variants in yeast genetic and yeast two-hybrid assays we show that the Zn-finger of Rad18 mediates its interaction with Rad5, and the N-terminal adjacent region is also necessary for Rad5 binding. Moreover, results of the yeast two-hybrid and in vivo ubiquitylation experiments raise the possibility that direct interaction between Rad18 and Rad5 might not be necessary for the function of the Rad5 dependent pathway. The presented data also reveal that yeast Rad18 uses different domains to mediate its association with itself and with Rad5. Our results contribute to better understanding of the complex machinery of DNA damage bypass pathways

Exploring the fitness benefits of genome reduction in Escherichia coli by a selection-driven approach

Artificial simplification of bacterial genomes is thought to have the potential to yield cells with reduced complexity, enhanced genetic stability, and improved cellular economy. Of these goals, economical gains, supposedly due to the elimination of superfluous genetic material, and manifested in elevated growth parameters in selected niches, have not yet been convincingly achieved. This failure might stem from limitations of the targeted genome reduction approach that assumes full knowledge of gene functions and interactions, and allows only a limited number of reduction trajectories to interrogate. To explore the potential fitness benefits of genome reduction, we generated successive random deletions in E. coli by a novel, selection-driven, iterative streamlining process. The approach allows the exploration of multiple streamlining trajectories, and growth periods inherent in the procedure ensure selection of the fittest variants of the population. By generating single- and multiple-deletion strains and reconstructing the deletions in the parental genetic background, we showed that favourable deletions can be obtained and accumulated by the procedure. The most reduced multiple-deletion strain, obtained in five deletion cycles (2.5% genome reduction), outcompeted the wild-type, and showed elevated biomass yield. The spectrum of advantageous deletions, however, affecting only a few genomic regions, appears to be limited

Enhancing the Translational Capacity of E. coli by Resolving the Codon Bias

Escherichia coli is a well-established and popular host for heterologous expression of proteins. The preference in the choice of synonymous codons (codon bias), however, might differ for the host and the original source of the recombinant protein, constituting a potential bottleneck in production. Codon choice affects the efficiency of translation by a complex and poorly understood mechanism. The availability of certain tRNA species is one of the factors that may curtail the capacity of translation. Here we provide a tRNA-overexpressing strategy that allows the resolution of the codon bias, and boosts the translational capacity of the popular host BL21(DE3) when rare codons are encountered. In the BL21(DE3)-derived strain, called SixPack, copies of the genes corresponding to the six least abundant tRNA species have been assembled in a synthetic fragment and inserted into a rRNA operon. This arrangement, while not interfering with the growth properties of the new strain, allows dynamic control of the transcription of the extra tRNA genes, providing significantly elevated levels of the rare tRNAs in the exponential growth phase. Results from expression assays of a panel of recombinant proteins of diverse origin and codon composition showed that the performance of SixPack surpassed that of the parental BL21(DE3) or a related strain equipped with a rare tRNA-expressing plasmid

Microbial genome engineering for promoting health and understanding disease

The completion of the first microbial genomes nearly two decades ago opened a completely new chapter in molecular genetics. The availability of precise sequence data permitted the extended use of existing genome engineering methods, and urged the development of a novel set of more rapid and simple techniques for genome editing. The rapidly decreasing price of sequencing and DNA synthesis opened further possibilities of high-throughput genetic analysis and assembly. As a consequence, biomedical knowledge increased at an exponential rate and accelerated the development of numerous connecting fields, including that of medical microbiology. This review presents the reader the toolbox available today to edit and assemble microbial genomes and showcases the key molecular genetic strategies employed to dissect the mechanisms of pathogenesis and construct microbial strains for preventive or therapeutic applications

Interactions between LPS moieties and macrophage pattern recognition receptors

Mammalian host organisms live their life constantly interacting with pathogenic and nonpathogenic Gram-negative bacteria. Commensal/symbiont strains are tolerated in the gut, while pathogens are kept at bay by the immune system. In contrast both commensals and pathogenic bacteria are targets of the immune system outside of the digestive system. Immune cells are activated upon contact with different constituents of bacterial cells like peptidoglycan, outer membrane proteins, fimbriae, bacterial DNA, etc. One of the dominant molecular targets affecting the immune cells is the lipopolysaccharide (LPS), an essential molecule of the cell wall of Gram-negative bacteria. In this review we discuss interactions of macrophages with the main LPS moieties lipid A, core and O-antigen regions

Capecitabine in Combination with Docetaxel in First Line in HER2-Negatíve Metastatic Breast Cancer: an Observational Study

Due to the limited experience with capecitabine plus docetaxel (XT) combination in the first-line treatment of metastatic breast cancer in Hungary, the main objective of the study was to analyze the effectiveness and tolerability of XT therapy. A prospective, open-label, non-randomized, single-arm, multicenter, observational study was designed. All female patients were eligible whose metastatic breast cancer could be treated with the XT protocol according to the summary of product characteristics of the drugs. The median progression free survival was 9.9 ± 3.0 months. Time to treatment failure was 4.6 ± 5.1 months on average. The overall response rate was 28.9 %, the clinical benefit rate was 73.3 %. The treatment was discontinued in 35.6 % of patients due to disease progression and in 20.0 % due to adverse events (AE). 33 patients with a total of 73 AEs have been reported, and 13 of them had serious adverse events (SAE). The efficacy and the safety profile of XT chemotherapy proven in the study are consistent with the results demonstrated in randomized trials. First-line XT chemotherapy effectively improves the PFS in metastatic breast cancer. Keywords CapecitabineDocetaxelHER2-negative metastatic breast cancerToxicit

Capecitabine in Combination with Docetaxel in First Line in HER2-Negative Metastatic Breast Cancer: an Observational Study

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Indispensability of Horizontally Transferred Genes and Its Impact on Bacterial Genome Streamlining

Why are certain bacterial genomes so small and compact? The adaptive genome streamlining hypothesis posits that selection acts to reduce genome size because of themetabolic burden of replicating DNA. To reveal the impact of genome streamlining on cellular traits, we reduced the Escherichia coli genome by up to 20% by deleting regions which have been repeatedly subjects of horizontal transfer in nature. Unexpectedly, horizontally transferred genes not only confer utilization of specific nutrients and elevate tolerance to stresses, but also allow efficient usage of resources to build new cells, and hence influence fitness in routine and stressful environments alike. Genome reduction affected fitness not only by gene loss, but also by induction of a general stress response. Finally, we failed to find evidence that the advantage of smaller genomes would be due to a reduced metabolic burden of replicating DNA or a link with smaller cell size. We conclude that as the potential energetic benefit gained by deletion of short genomic segments is vanishingly small compared with the deleterious side effects of these deletions, selection for reduced DNA synthesis costs is unlikely to shape the evolution of small genomes