Transcription regulation of the EcoRV restriction–modification system

When a plasmid containing restriction–modification (R–M) genes enters a naïve host, unmodified host DNA can be destroyed by restriction endonuclease. Therefore, expression of R–M genes must be regulated to ensure that enough methyltransferase is produced and that host DNA is methylated before the endonuclease synthesis begins. In several R–M systems, specialized Control (C) proteins coordinate expression of the R and the M genes. C proteins bind to DNA sequences called C-boxes and activate expression of their cognate R genes and inhibit the M gene expression, however the mechanisms remain undefined. Here, we studied the regulation of gene expression in the C protein-dependent EcoRV system. We map the divergent EcoRV M and R gene promoters and we define the site of C protein-binding that is sufficient for activation of the EcoRV R transcription

Type II restriction endonuclease R.Eco29kI is a member of the GIY-YIG nuclease superfamily

<p>Abstract</p> <p>Background</p> <p>The majority of experimentally determined crystal structures of Type II restriction endonucleases (REases) exhibit a common PD-(D/E)XK fold. Crystal structures have been also determined for single representatives of two other folds: PLD (R.BfiI) and half-pipe (R.PabI), and bioinformatics analyses supported by mutagenesis suggested that some REases belong to the HNH fold. Our previous bioinformatic analysis suggested that REase R.Eco29kI shares sequence similarities with one more unrelated nuclease superfamily, GIY-YIG, however so far no experimental data were available to support this prediction. The determination of a crystal structure of the GIY-YIG domain of homing endonuclease I-TevI provided a template for modeling of R.Eco29kI and prompted us to validate the model experimentally.</p> <p>Results</p> <p>Using protein fold-recognition methods we generated a new alignment between R.Eco29kI and I-TevI, which suggested a reassignment of one of the putative catalytic residues. A theoretical model of R.Eco29kI was constructed to illustrate its predicted three-dimensional fold and organization of the active site, comprising amino acid residues Y49, Y76, R104, H108, E142, and N154. A series of mutants was constructed to generate amino acid substitutions of selected residues (Y49A, R104A, H108F, E142A and N154L) and the mutant proteins were examined for their ability to bind the DNA containing the Eco29kI site 5'-CCGCGG-3' and to catalyze the cleavage reaction. Experimental data reveal that residues Y49, R104, E142, H108, and N154 are important for the nuclease activity of R.Eco29kI, while H108 and N154 are also important for specific DNA binding by this enzyme.</p> <p>Conclusion</p> <p>Substitutions of residues Y49, R104, H108, E142 and N154 predicted by the model to be a part of the active site lead to mutant proteins with strong defects in the REase activity. These results are in very good agreement with the structural model presented in this work and with our prediction that R.Eco29kI belongs to the GIY-YIG superfamily of nucleases. Our study provides the first experimental evidence for a Type IIP REase that does not belong to the PD-(D/E)XK or HNH superfamilies of nucleases, and is instead a member of the unrelated GIY-YIG superfamily.</p

Regulation of gene expression in restriction-modification system Eco29kI

The Eco29kI restriction-modification (R-M) system consists of two partially overlapping genes, eco29kIR, encoding a restriction endonuclease and eco29kIM, encoding methyltransferase. The two genes are thought to form an operon with the eco29kIR gene preceding the eco29kIM gene. Such an organization is expected to complicate establishment of plasmids containing this R-M system in naive hosts, since common logic dictates that methyltransferase should be synthesized first to protect the DNA from cleavage by the endonuclease. Here, we characterize the Eco29kI gene transcription. We show that a separate promoter located within the eco29kIR gene is sufficient to synthesize enough methyltransferase to completely modify host DNA. We further show that transcription from two intragenic antisense promoters strongly decreases the levels of eco29kIR gene transcripts. The antisense transcripts act by preventing translation initiation from the bicistronic eco29kIR–eco29kIM mRNA and causing its degradation. Both eco29kIM and antisense promoters are necessary for Eco29kI genes establishment and/or stable maintenance, indicating that they jointly contribute to coordinated expression of Eco29kI genes

Fused eco29kIR- and M genes coding for a fully functional hybrid polypeptide as a model of molecular evolution of restriction-modification systems

<p>Abstract</p> <p>Background</p> <p>The discovery of restriction endonucleases and modification DNA methyltransferases, key instruments of genetic engineering, opened a new era of molecular biology through development of the recombinant DNA technology. Today, the number of potential proteins assigned to type II restriction enzymes alone is beyond 6000, which probably reflects the high diversity of evolutionary pathways. Here we present experimental evidence that a new type IIC restriction and modification enzymes carrying both activities in a single polypeptide could result from fusion of the appropriate genes from preexisting bipartite restriction-modification systems.</p> <p>Results</p> <p>Fusion of <it>eco29kIR </it>and <it>M </it>ORFs gave a novel gene encoding for a fully functional hybrid polypeptide that carried both restriction endonuclease and DNA methyltransferase activities. It has been placed into a subclass of type II restriction and modification enzymes - type IIC. Its MTase activity, 80% that of the M.Eco29kI enzyme, remained almost unchanged, while its REase activity decreased by three times, concurrently with changed reaction optima, which presumably can be caused by increased steric hindrance in interaction with the substrate. <it>In vitro </it>the enzyme preferentially cuts DNA, with only a low level of DNA modification detected. <it>In vivo </it>new RMS can provide a 10²-fold less protection of host cells against phage invasion.</p> <p>Conclusions</p> <p>We propose a molecular mechanism of appearing of type IIC restriction-modification and M.SsoII-related enzymes, as well as other multifunctional proteins. As shown, gene fusion could play an important role in evolution of restriction-modification systems and be responsible for the enzyme subclass interconversion. Based on the proposed approach, hundreds of new type IIC enzymes can be generated using head-to-tail oriented type I, II, and III restriction and modification genes. These bifunctional polypeptides can serve a basis for enzymes with altered recognition specificities. Lastly, this study demonstrates that protein fusion may change biochemical properties of the involved enzymes, thus giving a starting point for their further evolutionary divergence.</p

Heart rate variability in workers of various professions in contrasting seasons of the year

Objectives: It is known that professional occupation affects the heart rate variability (HRV). However, most studies have not taken into account seasonal features of the HRV. The aim of this study has been to evaluate the HRV differences in winter and in summer in the case of the Ministry of the Russian Federation for Civil Defense, Emergencies and Elimination of Consequences of Natural Disasters (EMERCOM) workers and scientific workers from the Komi Science Center of the Ural Branch of the Russian Academy of Sciences. Material and Methods: The short-term HRV was examined for 13 EMERCOM workers and 13 scientific workers. The data was collected in winter (December) and summer (July) for the same groups of workers. The time domain and frequency domain HRV analyses were performed. The EMERCOM workers had more contact with the external environment than the scientific workers. Results: The two-way analysis of variance with repeated observations on a single factor has shown that “Season” and interaction of two factors “Season” and “Profession” significantly influenced the HRV among volunteers. The “Profession” factor did not influence the HRV parameters (except for the heart rate in winter, p = 0.042). Seasonal changes in the HRV parameters were not significant in the case of scientific workers. In contrast, the EMERCOM workers showed significantly decreased parameters of parasympathetic activity (the root-mean-square of successive differences in RR intervals, percentage of consecutive RR intervals differing by > 50 ms and the relative value high frequency power, p = 0.001, p = 0.014 and p = 0.009, respectively) and increased parameters of sympathetic activity (the stress index and ratio of low-frequency power to high-frequency power, p = 0.012 and p = 0.006, respectively) in winter as compared to summer. Conclusions: The results of our study indicate that, unlike the scientific workers, the EMERCOM workers showed significant changes in the HRV in contrasting seasons (winter and summer). A season of a year should be considered when assessing the HRV among workers of various professions. Int J Occup Med Environ Health 2016;29(5):793–80

Heart rate variability in workers of various professions in contrasting seasons of the year

Objectives: It is known that professional occupation affects the heart rate variability (HRV). However, most studies have not taken into account seasonal features of the HRV. The aim of this study has been to evaluate the HRV differences in winter and in summer in the case of the Ministry of the Russian Federation for Civil Defense, Emergencies and Elimination of Consequences of Natural Disasters (EMERCOM) workers and scientific workers from the Komi Science Center of the Ural Branch of the Russian Academy of Sciences. Material and Methods: The short-term HRV was examined for 13 EMERCOM workers and 13 scientific workers. The data was collected in winter (December) and summer (July) for the same groups of workers. The time domain and frequency domain HRV analyses were performed. The EMERCOM workers had more contact with the external environment than the scientific workers. Results: The two-way analysis of variance with repeated observations on a single factor has shown that “Season” and interaction of two factors “Season” and “Profession” significantly influenced the HRV among volunteers. The “Profession” factor did not influence the HRV parameters (except for the heart rate in winter, p = 0.042). Seasonal changes in the HRV parameters were not significant in the case of scientific workers. In contrast, the EMERCOM workers showed significantly decreased parameters of parasympathetic activity (the root-mean-square of successive differences in RR intervals, percentage of consecutive RR intervals differing by > 50 ms and the relative value high frequency power, p = 0.001, p = 0.014 and p = 0.009, respectively) and increased parameters of sympathetic activity (the stress index and ratio of low-frequency power to high-frequency power, p = 0.012 and p = 0.006, respectively) in winter as compared to summer. Conclusions: The results of our study indicate that, unlike the scientific workers, the EMERCOM workers showed significant changes in the HRV in contrasting seasons (winter and summer). A season of a year should be considered when assessing the HRV among workers of various professions. Int J Occup Med Environ Health 2016;29(5):793–80