Although restriction-modification systems are found in both Eubacterial and Archaeal kingdoms, comparatively less is known about patterns of DNA methylation and genome defense systems in archaea. ...Here we report the complete closed genome sequence and methylome analysis of
Methanococcus aeolicus
PL15/H
p
, a strain of the CO
2
-reducing methanogenic archaeon and a commercial source for
Mae
I,
Mae
II, and
Mae
III restriction endonucleases. The
M. aeolicus
PL15/H
p
genome consists of a 1.68 megabase circular chromosome predicted to contain 1,615 protein coding genes and 38 tRNAs. A combination of methylome sequencing, homology-based genome annotation, and recombinant gene expression identified five restriction-modification systems encoded by this organism, including the methyltransferase and site-specific endonuclease of
Mae
III. The
Mae
III restriction endonuclease was recombinantly expressed, purified and shown to have site-specific DNA cleavage activity
in vitro
.
Two strains of filamentous, colorless sulfur bacteria were isolated from bacterial fouling in the outflow of hydrogen sulfide-containing waters from a coal mine (
Thiothrix
sp. Ku-5) and on the ...seashore of the White Sea (
Thiothrix
sp. AS). Metagenome-assembled genome (MAG) A52 was obtained from a sulfidic spring in the Volgograd region, Russia. Phylogenetic analysis based on the 16S rRNA gene sequences showed that all genomes represented the genus
Thiothrix
. Based on their average nucleotide identity and digital DNA-DNA hybridization data these new isolates and the MAG represent three species within the genus
Thiothrix
with the proposed names
Thiothrix subterranea
sp. nov. Ku-5
T
,
Thiothrix litoralis
sp. nov. AS
T
, and “
Candidatus
Thiothrix anitrata” sp. nov. A52. The complete genome sequences of
Thiothrix fructosivorans
Q
T
and
Thiothrix unzii
A1
T
were determined. Complete genomes of seven
Thiothrix
isolates, as well as two MAGs, were used for pangenome analysis. The
Thiothrix
core genome consisted of 1,355 genes, including ones for the glycolysis, the tricarboxylic acid cycle, the aerobic respiratory chain, and the Calvin cycle of carbon fixation. Genes for dissimilatory oxidation of reduced sulfur compounds, namely the branched SOX system (
SoxAXBYZ
), direct (
soeABC
) and indirect (
aprAB
,
sat
) pathways of sulfite oxidation, sulfur oxidation complex Dsr (
dsrABEFHCEMKLJONR
), sulfide oxidation systems SQR (
sqrA
,
sqrF
), and FCSD (
fccAB
) were found in the core genome. Genomes differ in the set of genes for dissimilatory reduction of nitrogen compounds, nitrogen fixation, and the presence of various types of RuBisCO.
Oxidation of nanosized titanium (nano-Ti), a promising component of energetic compounds, was studied using thermogravimetry and differential scanning calorimetry. To obtain more comprehensive insight ...into the kinetics and mechanism of oxidation, a variety of complementary non-isothermal and isothermal thermoanalytical experiments were performed. In sharp contrast to micron-sized titanium, oxidation of nano-Ti commences at much lower temperatures (150 °C instead of 650 °C) with profoundly lower activation energies (152 ± 3 kJ mol−1 and 220 ± 3 kJ mol−1, respectively). Moreover, reaction kinetics for nano-Ti obeys the logarithmic law, while in the case of micron-sized Ti kinetics is described by the 2D-diffusion model. At the microscopic level, the observed kinetics of nano-Ti oxidation is explained by switching of the limiting reaction stage to short-circuit diffusion of oxygen through the titanium oxide. This process is promoted by the increase of porosity upon initial water loss and the blocking of pores in the course of oxidation. The kinetic model proposed for oxidation of nano-Ti was independently benchmarked against the isothermal kinetics (zero heating rate limit) and ignition data (high heating rates). Our model provides reliable kinetics of the nano-Ti oxidation, which is valid for both storage and application conditions.
Graphical Abstract
Cartoon illustration part of this work (Human ACE2 N-terminal domain tethered to RNase A and RNA degradation by the fusion enzyme).
The SARS-CoV-2 viral genome contains a ...positive-strand single-stranded RNA of ∼30 kb. Human ACE2 protein is the receptor for SARS-CoV-2 virus attachment and infection. We propose to use ribonucleases (RNases) as antiviral agents to destroy the viral genome
in vitro
. In the virions, the RNA is protected by viral capsid proteins, membrane proteins, and nucleocapsid proteins. To utilize RNases as antiviral strategy, we set out to construct RNase fusion with human ACE2 receptor N-terminal domain (ACE2NTD). We expressed six proteins in
E. coli
cells: (1) MBP-ACE2NTD, (2) ACE2NTD-GFP, (3) RNase I (6×His), (4) RNase III (6×His), (5) RNase I-ACE2NTD (6×His), and (6) human RNase A-ACE2NTD (6×His). We evaluated fusion expression in different
E. coli
strains, partially purified MBP-ACE2NTD protein from the soluble fraction of bacterial cell lysate, and refolded MBP-ACE2NTD protein from inclusion body. The engineered RNase I-ACE2NTD (6×His) and hRNase A-ACE2NTD (6×His) fusions are active in cleaving SARS-CoV-2 RNA fragment
in vitro
. The recombinant RNase I (6×His) and RNase III (6×His) are active in cleaving RNA and dsRNA in test tube. This study provides a proof-of-concept for construction of fusion protein between human receptor and nuclease that may be used to degrade viral nucleic acids.
P53 family members with a transactivation domain induce cell cycle arrest and promoteapoptosis. However, ΔNp63 isotypes lacking the transactivation (TA)- domain promote cellproliferation and ...tumorigenesis in vitro and in vivo. Although p53, TAp63 or TAp73 are stabilizedupon DNA damage, we found that the genotoxic stress agents induced a dramatic decrease andphosphorylation of ΔNp63α in squamous cell carcinoma cells. Further work revealed that RACK1physically associated with the p63α C-terminal domain through its WD40 domain. However,stratifin binds with phosphorylated ΔNp63α in response to cisplatin. Upon DNA damage inducedby cisplatin, stratifin mediated a nuclear export of ΔNp63α into cytoplasm and then RACK1targeted latter into a proteasome degradation pathway possibly serving as an E3 ubiquitin ligase.Moreover, siRNA knockdown of both stratifin and RACK1 inhibited a nuclear export and proteindegradation of ΔNp63α, respectively. Our data suggest that modification and down regulation ofΔNp63α is one of the major determinants of the cellular response to DNA damage in human headand neck cancers.
We report a new subgroup of Type III Restriction-Modification systems that use m4C methylation for host protection. Recognition specificities for six such systems, each recognizing a novel motif, ...have been determined using single molecule real-time DNA sequencing. In contrast to all previously characterized Type III systems which modify adenine to m6A, protective methylation of the host genome in these new systems is achieved by the N4-methylation of a cytosine base in one strand of an asymmetric 4 to 6 base pair recognition motif. Type III systems are heterotrimeric enzyme complexes containing a single copy of an ATP-dependent restriction endonuclease-helicase (Res) and a dimeric DNA methyltransferase (Mod). The Type III Mods are beta-class amino-methyltransferases, examples of which form either N6-methyl adenine or N4-methyl cytosine in Type II RM systems. The Type III m4C Mod and Res proteins are diverged, suggesting ancient origin or that m4C modification has arisen from m6A MTases multiple times in diverged lineages. Two of the systems, from thermophilic organisms, required expression of both Mod and Res to efficiently methylate an E. coli host, unlike previous findings that Mod alone is proficient at modification, suggesting that the division of labor between protective methylation and restriction activities is atypical in these systems. Two of the characterized systems, and many homologous putative systems, appear to include a third protein; a conserved putative helicase/ATPase subunit of unknown function and located 5' of the mod gene. The function of this additional ATPase is not yet known, but close homologs co-localize with the typical Mod and Res genes in hundreds of putative Type III systems. Our findings demonstrate a rich diversity within Type III RM systems.
Celotno besedilo
Dostopno za:
DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
Here, we report a novel endonuclease and N6-adenine DNA methyltransferase (m6A methyltransferase) in the Ureaplasma parvum SV3F4 strain. Our previous study found that the SV3F4 strain carries 17 ...unique genes, which are not encoded in the two previously reported U. parvum serovar 3 strain, OMC-P162 and ATCC 700970. Of these 17 unique genes, UP3_c0261 and UP3_c0262, were originally annotated as encoding hypothetical proteins. Comparative genomics analyses more recently indicated they encode a Type II restriction endonuclease and an m6A methyltransferase, respectively. The UP3_c0261 and UP3_c0262 genes were individually expressed and purified in Escherichia coli. The UP3_c0261 recombinant protein showed endonuclease activity on the pT7Blue vector, recognizing and cleaving a GTNAC motif, resulting in a 5 base 5’ extension. The UP3_c0261 protein digested a polymerase chain reaction (PCR) product harboring the GTNAC motif. The endonuclease UP3_c0261 was designated as UpaF4I. Treatment of the PCR product with the recombinant protein UP3_c0262 completely blocked the restriction enzyme activity of UpaF4I. Analysis of the treated PCR product harboring a modified nucleotide by UP3_c0262 with HPLC-MS/MS and MS/MS showed that UP3_c0262 was an m6A methyltransferase containing a methylated A residue in both DNA strands of the GTNAC motif. Whole genome methylation analysis of SV3F4 showed that 99.9 % of the GTNAC motif was m6A modified. These results suggest the UP3_c0261 and UP3_c0262 genes may act as a novel Type II restriction-modification system in the Ureaplasma SV3F4 strain.
•UP3_c0261 protein is a Type II endonuclease, designated as UpaF4I.•UpaF4I recognized the ↓GTNAC↑ motif, resulting in a 5 base 5’ extension.•UP3_c0262 completely blocked UpaF4I restriction enzyme activity.•UP3_c0262 protein (M.UpaF4I) is an m6A DNA methyltransferase.
MspJI is a novel modification-dependent restriction endonuclease that cleaves at a fixed distance away from the modification site. Here, we present the biochemical characterization of several MspJI ...homologs, including FspEI, LpnPI, AspBHI, RlaI, and SgrTI. All of the enzymes specifically recognize cytosine C5 modification (methylation or hydroxymethylation) in DNA and cleave at a constant distance (N12/N16) away from the modified cytosine. Each displays its own sequence context preference, favoring different nucleotides flanking the modified cytosine. By cleaving on both sides of fully modified CpG sites, they allow the extraction of 32-base long fragments around the modified sites from the genomic DNA. These enzymes provide powerful tools for direct interrogation of the epigenome. For example, we show that RlaI, an enzyme that prefers mCWG but not mCpG sites, generates digestion patterns that differ between plant and mammalian genomic DNA, highlighting the difference between their epigenomic patterns. In addition, we demonstrate that deep sequencing of the digested DNA fragments generated from these enzymes provides a feasible method to map the modified sites in the genome. Altogether, the MspJI family of enzymes represent appealing tools of choice for method development in DNA epigenetic studies.
Prevotella intermedia, a major periodontal pathogen, is increasingly implicated in human respiratory tract and cystic fibrosis lung infections. Nevertheless, the specific mechanisms employed by this ...pathogen remain only partially characterized and poorly understood, largely due to its total lack of genetic accessibility. Here, using Single Molecule, Real-Time (SMRT) genome and methylome sequencing, bisulfite sequencing, in addition to cloning and restriction analysis, we define the specific genetic barriers to exogenous DNA present in two of the most widespread laboratory strains, P. intermedia ATCC 25611 and P. intermedia Strain 17. We identified and characterized multiple restriction-modification (R-M) systems, some of which are considerably divergent between the two strains. We propose that these R-M systems are the root cause of the P. intermedia transformation barrier. Additionally, we note the presence of conserved Clustered Regularly Interspaced Short Palindromic Repeat (CRISPR) systems in both strains, which could provide a further barrier to exogenous DNA uptake and incorporation. This work will provide a valuable resource during the development of a genetic system for P. intermedia, which will be required for fundamental investigation of this organism's physiology, metabolism, and pathogenesis in human disease.
Celotno besedilo
Dostopno za:
DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
Although restriction-modification systems are found in both Eubacterial and Archaeal kingdoms, comparatively less is known about patterns of DNA methylation and genome defense systems in archaea. ...Here we report the complete closed genome sequence and methylome analysis of
PL15/H
, a strain of the CO
-reducing methanogenic archaeon and a commercial source for
I,
II, and
III restriction endonucleases. The
PL15/H
genome consists of a 1.68 megabase circular chromosome predicted to contain 1,615 protein coding genes and 38 tRNAs. A combination of methylome sequencing, homology-based genome annotation, and recombinant gene expression identified five restriction-modification systems encoded by this organism, including the methyltransferase and site-specific endonuclease of
III. The
III restriction endonuclease was recombinantly expressed, purified and shown to have site-specific DNA cleavage activity
.
PDF
