Complete genome sequence of new Microbacterium sp. strain ET2, isolated from roots of leafless orchid.

Whole-genome sequence of ET2 strain, isolated from the roots of leafless orchid, constitutes a single circular chromosome of 3,604,840 bp (69.44% G + C content). BLAST+-based average nucleotide identity (ANIb) and digital DNA-DNA hybridization values indicate that ET2 may be a novel Microbacterium species. Genes putatively involved in plant-microbial interactions were predicted.

Conserved G-Quadruplex-Forming Sequences in Mammalian TERT Promoters and Their Effect on Mutation Frequency.

Somatic mutations in the promoter region of the human telomerase reverse transcriptase (hTERT) gene have been identified in many types of cancer. The hTERT promoter is known to be enriched with sequences that enable the formation of G-quadruplex (G4) structures, whose presence is associated with elevated mutagenicity and genome instability. Here, we used a bioinformatics tool (QGRS mapper) to search for G4-forming sequences (G4 motifs) in the 1000 bp TERT promoter regions of 141 mammalian species belonging to 20 orders, 5 of which, including primates and predators, contain more than 10 species. Groups of conserved G4 motifs and single-nucleotide variants within these groups were discovered using a block alignment approach (based on the Nucleotide PanGenome explorer). It has been shown that: (i) G4 motifs are predominantly located in the region proximal to the transcription start site (up to 400 bp) and are over-represented on the non-coding strand of the TERT promoters, (ii) 11 to 22% of the G4 motifs found are evolutionarily conserved across the related organisms, and (iii) a statistically significant higher frequency of nucleotide substitutions in the conserved G4 motifs compared to the surrounding regions was confirmed only for the order Primates. These data support the assumption that G4s can interfere with the DNA repair process and affect the evolutionary adaptation of organisms and species.

Draft Genome Sequence of Thermomicrobium sp. Strain 4228-Ro, a Thermophilic Bacterium Isolated from a Kamchatka Hot Spring.

The genome of Thermomicrobium sp. strain 4228-Ro, an aerobic thermophilic bacterium isolated from a Kamchatka hot spring, was sequenced and analyzed. The genome assembly comprises 13 contigs with a total length of 3,068,448 bp. Genome analysis revealed the pathway of aerobic utilization of sugars, which was corroborated by growth experiments.

New DNA Plasmid Model for Studying DNA Mismatch Repair Response to the G4 Structure.

G-quadruplexes (G4s), the most widely studied alternative DNA structures, are implicated in the regulation of the key cellular processes. In recent years, their involvement in DNA repair machinery has become the subject of intense research. Here, we evaluated the effect of G4 on the prokaryotic DNA mismatch repair (MMR) pathway from two bacterial sources with different mismatch repair mechanisms. The G4 folding, which competes with the maintenance of double-stranded DNA, is known to be controlled by numerous opposing factors. To overcome the kinetic barrier of G4 formation, we stabilized a parallel G4 formed by the d(GGGT)4 sequence in a DNA plasmid lacking a fragment complementary to the G4 motif. Unlike commonly used isolated G4 structures, our plasmid with an embedded stable G4 structure contained elements, such as a MutH cleavage site, required to initiate the repair process. G4 formation in the designed construct was confirmed by Taq polymerase stop assay and dimethyl sulfate probing. The G4-carrying plasmid, together with control ones (lacking a looped area or containing unstructured d(GT)8 insert instead of the G4 motif), were used as new type models to answer the question of whether G4 formation interferes with DNA cleavage as a basic function of MMR.

Model of the SARS-CoV-2 Virus for Development of a DNA-Modified, Surface-Enhanced Raman Spectroscopy Sensor with a Novel Hybrid Plasmonic Platform in Sandwich Mode.

The recent severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection has posed a great challenge for the development of ultra-fast methods for virus identification based on sensor principles. We created a structure modeling surface and size of the SARS-CoV-2 virus and used it in comparison with the standard antigen SARS-CoV-2-the receptor-binding domain (RBD) of the S-protein of the envelope of the SARS-CoV-2 virus from the Wuhan strain-for the development of detection of coronaviruses using a DNA-modified, surface-enhanced Raman scattering (SERS)-based aptasensor in sandwich mode: a primary aptamer attached to the plasmonic surface-RBD-covered Ag nanoparticle-the Cy3-labeled secondary aptamer. Fabricated novel hybrid plasmonic structures based on "Ag mirror-SiO2-nanostructured Ag" demonstrate sensitivity for the detection of investigated analytes due to the combination of localized surface plasmons in nanostructured silver surface and the gap surface plasmons in a thin dielectric layer of SiO2 between silver layers. A specific SERS signal has been obtained from SERS-active compounds with RBD-specific DNA aptamers that selectively bind to the S protein of synthetic virion (dissociation constants of DNA-aptamer complexes with protein in the range of 10 nM). The purpose of the study is to systematically analyze the combination of components in an aptamer-based sandwich system. A developed virus size simulating silver particles adsorbed on an aptamer-coated sensor provided a signal different from free RBD. The data obtained are consistent with the theory of signal amplification depending on the distance of the active compound from the amplifying surface and the nature of such a compound. The ability to detect the target virus due to specific interaction with such DNA is quantitatively controlled by the degree of the quenching SERS signal from the labeled compound. Developed indicator sandwich-type systems demonstrate high stability. Such a platform does not require special permissions to work with viruses. Therefore, our approach creates the promising basis for fostering the practical application of ultra-fast, amplification-free methods for detecting coronaviruses based on SARS-CoV-2.

G-Quadruplex Formed by the Promoter Region of the hTERT Gene: Structure-Driven Effects on DNA Mismatch Repair Functions.

G-quadruplexes (G4s) are a unique class of noncanonical DNAs that play a key role in cellular processes and neoplastic transformation. Herein, we focused on the promoter region of human TERT oncogene, whose product is responsible for the immortality of cancer cells. It has been shown by chemical probing and spectroscopic methods that synthetic 96-nt DNAs modeling the wild-type G-rich strand of the hTERT promoter and its variants with G>A point substitutions corresponding to somatic driver mutations fold into three stacked parallel G4s with sites of local G4 destabilization caused by G>A substitutions in the G4 motif. These models were used to elucidate how the hTERT multiG4 affects the binding affinity and functional responses of two key proteins, MutS and MutL, involved in the initial stage of DNA mismatch repair (MMR) in Escherichiacoli and Neisseriagonorrhoeae with different MMR mechanisms. We have shown for the first time that (i) point substitutions do not affect the effective binding of these proteins to the hTERT G4 structure, and (ii) the endonuclease activity of MutL from N. gonorrhoeae is significantly suppressed by the stable G4 scaffold. It is likely that some of the genomic instability associated with G4 may be related to the blockage of human intrinsic methyl-independent MMR attempting to operate near G4 structures.

Nanopore Sequencing for De Novo Bacterial Genome Assembly and Search for Single-Nucleotide Polymorphism.

Nanopore sequencing (ONT) is a new and rapidly developing method for determining nucleotide sequences in DNA and RNA. It serves the ability to obtain long reads of thousands of nucleotides without assembly and amplification during sequencing compared to next-generation sequencing. Nanopore sequencing can help for determination of genetic changes leading to antibiotics resistance. This study presents the application of ONT technology in the assembly of an E. coli genome characterized by a deletion of the tolC gene and known single-nucleotide variations leading to antibiotic resistance, in the absence of a reference genome. We performed benchmark studies to determine minimum coverage depth to obtain a complete genome, depending on the quality of the ONT data. A comparison of existing programs was carried out. It was shown that the Flye program demonstrates plausible assembly results relative to others (Shasta, Canu, and Necat). The required coverage depth for successful assembly strongly depends on the size of reads. When using high-quality samples with an average read length of 8 Kbp or more, the coverage depth of 30× is sufficient to assemble the complete genome de novo and reliably determine single-nucleotide variations in it. For samples with shorter reads with mean lengths of 2 Kbp, a higher coverage depth of 50× is required. Avoiding of mechanical mixing is obligatory for samples preparation. Nanopore sequencing can be used alone to determine antibiotics-resistant genetic features of bacterial strains.

Identification of a Novel Small RNA Encoded in the Mouse Urokinase Receptor uPAR Gene (Plaur) and Its Molecular Target Mef2d.

Urokinase receptor (uPAR) is a glycosylphosphatidylinositol (GPI)-anchored receptor of urokinase (uPA), which is involved in brain development, nerve regeneration, wound healing and tissue remodeling. We have recently shown that Plaur, which encodes uPAR, is an early response gene in murine brain. Assumingly, diverse functions of Plaur might be attributed to hypothetical, unidentified microRNAs encoded within introns of the Plaur gene. Using a bioinformatic approach we identified novel small RNAs within the Plaur gene and named them Plaur-miR1-3p and Plaur-miR1-5p. We confirmed Plaur-dependent expression of Plaur-miR1-3p and Plaur-miR1-5p in the mouse brain and mouse neuroblastoma Neuro2a cells. Utilizing an in silico MR-microT algorithm in DianaTools we selected two target genes - Mef2d and Emx2 with the highest binding scores to small RNAs selected from identified Plaur-Pre-miR1. Furthermore, sequencing of mouse brain samples for Plaur-miR1-5p target genes revealed two more genes-Nrip3 and Snrnp200. The expression of Emx2, Mef2d, and Snrnp200 in the mouse brain and Mef2d and Snrnp200 in Neuro2a cells correlated with expression of Plaur and small RNAs-Plaur-miR1-3p and Plaur-miR1-5p. Finally, we demonstrated elevated MEF2D protein expression in the mouse brain after Plaur induction and displayed activating effects of Plaur-miR1-5p on Mef2d expression in Neuro2a cells using Luciferase reporter assay. In conclusion, we have identified Plaur-miR1-3p and Plaur-miR1-5p as novel small RNAs encoded in the Plaur gene. This finding expands the current understanding of Plaur function in brain development and functioning.

Reactive Acrylamide-Modified DNA Traps for Accurate Cross-Linking with Cysteine Residues in DNA-Protein Complexes Using Mismatch Repair Protein MutS as a Model.

Covalent protein capture (cross-linking) by reactive DNA derivatives makes it possible to investigate structural features by fixing complexes at different stages of DNA-protein recognition. The most common cross-linking methods are based on reactive groups that interact with native or engineered cysteine residues. Nonetheless, high reactivity of most of such groups leads to preferential fixation of early-stage complexes or even non-selective cross-linking. We synthesised a set of DNA reagents carrying an acrylamide group attached to the C5 atom of a 2'-deoxyuridine moiety via various linkers and studied cross-linking with MutS as a model protein. MutS scans DNA for mismatches and damaged nucleobases and can form multiple non-specific complexes with DNA that may cause non-selective cross-linking. By varying the length of the linker between DNA and the acrylamide group and by changing the distance between the reactive nucleotide and a mismatch in the duplex, we showed that cross-linking occurs only if the distance between the acrylamide group and cysteine is optimal within the DNA-protein complex. Thus, acrylamide-modified DNA duplexes are excellent tools for studying DNA-protein interactions because of high selectivity of cysteine trapping.

Telomere length regulation by Rif1 protein from Hansenula polymorpha.

Rif1 is a large multifaceted protein involved in various processes of DNA metabolism - from telomere length regulation and replication to double-strand break repair. The mechanistic details of its action, however, are often poorly understood. Here, we report functional characterization of the Rif1 homologue from methylotrophic thermotolerant budding yeast Hansenula polymorpha DL-1. We show that, similar to other yeast species, H. polymorpha Rif1 suppresses telomerase-dependent telomere elongation. We uncover two novel modes of Rif1 recruitment at H. polymorpha telomeres: via direct DNA binding and through the association with the Ku heterodimer. Both of these modes (at least partially) require the intrinsically disordered N-terminal extension - a region of the protein present exclusively in yeast species. We also demonstrate that Rif1 binds Stn1 and promotes its accumulation at telomeres in H. polymorpha.

Impact of G-Quadruplexes on the Regulation of Genome Integrity, DNA Damage and Repair.

DNA G-quadruplexes (G4s) are known to be an integral part of the complex regulatory systems in both normal and pathological cells. At the same time, the ability of G4s to impede DNA replication plays a critical role in genome integrity. This review summarizes the results of recent studies of G4-mediated genomic and epigenomic instability, together with associated DNA damage and repair processes. Although the underlying mechanisms remain to be elucidated, it is known that, among the proteins that recognize G4 structures, many are linked to DNA repair. We analyzed the possible role of G4s in promoting double-strand DNA breaks, one of the most deleterious DNA lesions, and their repair via error-prone mechanisms. The patterns of G4 damage, with a focus on the introduction of oxidative guanine lesions, as well as their removal from G4 structures by canonical repair pathways, were also discussed together with the effects of G4s on the repair machinery. According to recent findings, there must be a delicate balance between G4-induced genome instability and G4-promoted repair processes. A broad overview of the factors that modulate the stability of G4 structures in vitro and in vivo is also provided here.

Responses of DNA Mismatch Repair Proteins to a Stable G-Quadruplex Embedded into a DNA Duplex Structure.

DNA mismatch repair (MMR) plays a crucial role in the maintenance of genomic stability. The main MMR protein, MutS, was recently shown to recognize the G-quadruplex (G4) DNA structures, which, along with regulatory functions, have a negative impact on genome integrity. Here, we studied the effect of G4 on the DNA-binding activity of MutS from Rhodobacter sphaeroides (methyl-independent MMR) in comparison with MutS from Escherichia coli (methyl-directed MMR) and evaluated the influence of a G4 on the functioning of other proteins involved in the initial steps of MMR. For this purpose, a new DNA construct was designed containing a biologically relevant intramolecular stable G4 structure flanked by double-stranded regions with the set of DNA sites required for MMR initiation. The secondary structure of this model was examined using NMR spectroscopy, chemical probing, fluorescent indicators, circular dichroism, and UV spectroscopy. The results unambiguously showed that the d(GGGT)4 motif, when embedded in a double-stranded context, adopts a G4 structure of a parallel topology. Despite strong binding affinities of MutS and MutL for a G4, the latter is not recognized by E. coli MMR as a signal for repair, but does not prevent MMR processing when a G4 and G/T mismatch are in close proximity.

Insights into the structure and function of Est3 from the Hansenula polymorpha telomerase.

Telomerase is a ribonucleoprotein enzyme, which maintains genome integrity in eukaryotes and ensures continuous cellular proliferation. Telomerase holoenzyme from the thermotolerant yeast Hansenula polymorpha, in addition to the catalytic subunit (TERT) and telomerase RNA (TER), contains accessory proteins Est1 and Est3, which are essential for in vivo telomerase function. Here we report the high-resolution structure of Est3 from Hansenula polymorpha (HpEst3) in solution, as well as the characterization of its functional relationships with other components of telomerase. The overall structure of HpEst3 is similar to that of Est3 from Saccharomyces cerevisiae and human TPP1. We have shown that telomerase activity in H. polymorpha relies on both Est3 and Est1 proteins in a functionally symmetrical manner. The absence of either Est3 or Est1 prevents formation of a stable ribonucleoprotein complex, weakens binding of a second protein to TER, and decreases the amount of cellular TERT, presumably due to the destabilization of telomerase RNP. NMR probing has shown no direct in vitro interactions of free Est3 either with the N-terminal domain of TERT or with DNA or RNA fragments mimicking the probable telomerase environment. Our findings corroborate the idea that telomerase possesses the evolutionarily variable functionality within the conservative structural context.

Functional Analysis of DNMT3A DNA Methyltransferase Mutations Reported in Patients with Acute Myeloid Leukemia.

In mammals, DNA methylation is necessary for the maintenance of genomic stability, gene expression regulation, and other processes. During malignant diseases progression, changes in both DNA methylation patterns and DNA methyltransferase (MTase) genes are observed. Human de novo MTase DNMT3A is most frequently mutated in acute myeloid leukemia (AML) with a striking prevalence of R882H mutation, which has been extensively studied. Here, we investigate the functional role of the missense mutations (S714C, R635W, R736H, R771L, P777R, and F752V) found in the catalytic domain of DNMT3A in AML patients. These were accordingly mutated in the murine Dnmt3a catalytic domain (S124C, R45W, R146H, R181L, P187R, and F162V) and in addition, one-site CpG-containing DNA substrates were used as a model system. The 3-15-fold decrease (S124C and P187R) or complete loss (F162V, R45W, and R146H) of Dnmt3a-CD methylation activity was observed. Remarkably, Pro 187 and Arg 146 are not located at or near the Dnmt3a functional motives. Regulatory protein Dnmt3L did not enhance the methylation activity of R45W, R146H, P187R, and F162V mutants. The key steps of the Dnmt3a-mediated methylation mechanism, including DNA binding and transient covalent intermediate formation, were examined. There was a complete loss of DNA-binding affinity for R45W located in the AdoMet binding region and for R146H. Dnmt3a mutants studied in vitro suggest functional impairment of DNMT3A during pathogenesis.

Functional duplication of Rap1 in methylotrophic yeasts.

The telomere regulator and transcription factor Rap1 is the only telomere protein conserved in yeasts and mammals. Its functional repertoire in budding yeasts is a particularly interesting field for investigation, given the high evolutionary diversity of this group of unicellular organisms. In the methylotrophic thermotolerant species Hansenula polymorpha DL-1 the RAP1 gene is duplicated (HpRAP1A and HpRAP1B). Here, we report the functional characterization of the two paralogues from H. polymorpha DL-1. We uncover distinct (but overlapping) DNA binding preferences of HpRap1A and HpRap1B proteins. We show that only HpRap1B is able to recognize telomeric DNA directly and to protect it from excessive recombination, whereas HpRap1A is associated with subtelomere regions. Furthermore, we identify specific binding sites for both HpRap1A and HpRap1B within promoters of a large number of ribosomal protein genes (RPGs), implicating Rap1 in the control of the RP regulon in H. polymorpha. Our bioinformatic analysis suggests that RAP1 was duplicated early in the evolution of the "methylotrophs" clade, and the two genes evolved independently. Therefore, our characterization of Rap1 paralogues in H. polymorpha may be relevant to other "methylotrophs", yielding valuable insights into the evolution of budding yeasts.

Structure and function of the N-terminal domain of the yeast telomerase reverse transcriptase.

The elongation of single-stranded DNA repeats at the 3'-ends of chromosomes by telomerase is a key process in maintaining genome integrity in eukaryotes. Abnormal activation of telomerase leads to uncontrolled cell division, whereas its down-regulation is attributed to ageing and several pathologies related to early cell death. Telomerase function is based on the dynamic interactions of its catalytic subunit (TERT) with nucleic acids-telomerase RNA, telomeric DNA and the DNA/RNA heteroduplex. Here, we present the crystallographic and NMR structures of the N-terminal (TEN) domain of TERT from the thermotolerant yeast Hansenula polymorpha and demonstrate the structural conservation of the core motif in evolutionarily divergent organisms. We identify the TEN residues that are involved in interactions with the telomerase RNA and in the recognition of the 'fork' at the distal end of the DNA product/RNA template heteroduplex. We propose that the TEN domain assists telomerase biological function and is involved in restricting the size of the heteroduplex during telomere repeat synthesis.

Chemical shift assignments and the secondary structure of the Est3 telomerase subunit in the yeast Hansenula polymorpha.

Telomerase is a multisubunit ribonucleoprotein enzyme that is essential for continuous cellular proliferation. A key role of telomerase in cancer and ageing makes it a promising target for the development of cancer therapies and treatments of other age-associated diseases, since telomerase allows unlimited proliferation potential of cells in the majority of cancer types. However, the structure and molecular mechanism of telomerase action are still poorly understood. In budding yeast, telomerase consists of the catalytic subunit, the telomerase reverse transcriptase or Est2 protein, telomerase RNA (TLC1) and two regulatory subunits, Est1 and Est3. Each of the four subunits is essential for in vivo telomerase function. Est3 interacts directly with Est1 and Est2, and stimulates Est2 catalytic activity. However, the exact role of the Est3 protein in telomerase function is still unknown. Determination of the structure, dynamic and functional properties of Est3 can bring new insights into the molecular mechanism of telomerase activity. Here we report nearly complete 1H, 13C and 15N resonance assignments of Est3 from the yeast Hansenula polymorpha. Analysis of the assigned chemical shifts allowed us to identify the protein's secondary structure and backbone dynamic properties. Structure-based sequence alignment revealed similarities in the structural organization of yeast Est3 and mammalian TPP1 proteins.

The genome-wide transcription response to telomerase deficiency in the thermotolerant yeast Hansenula polymorpha DL-1.

BACKGROUND: In the course of replication of eukaryotic chromosomes, the telomere length is maintained due to activity of telomerase, the ribonucleoprotein reverse transcriptase. Abolishing telomerase function causes progressive shortening of telomeres and, ultimately, cell cycle arrest and replicative senescence. To better understand the cellular response to telomerase deficiency, we performed a transcriptomic study for the thermotolerant methylotrophic yeast Hansenula polymorpha DL-1 lacking telomerase activity. RESULTS: Mutant strain of H. polymorpha carrying a disrupted telomerase RNA gene was produced, grown to senescence and analyzed by RNA-seq along with wild type strain. Telomere shortening induced a transcriptional response involving genes relevant to telomere structure and maintenance, DNA damage response, information processing, and some metabolic pathways. Genes involved in DNA replication and repair, response to environmental stresses and intracellular traffic were up-regulated in senescent H. polymorpha cells, while strong down-regulation was observed for genes involved in transcription and translation, as well as core histones. CONCLUSIONS: Comparison of the telomerase deletion transcription responses by Saccharomyces cerevisiae and H. polymorpha demonstrates that senescence makes different impact on the main metabolic pathways of these yeast species but induces similar changes in processes related to nucleic acids metabolism and protein synthesis. Up-regulation of a subunit of the TORC1 complex is clearly relevant for both types of yeast.

NMR assignments of the N-terminal domain of Ogataea polymorpha telomerase reverse transcriptase.

Telomerase is a ribonucleoprotein enzyme that adds telomeric DNA fragments to the ends of chromosomes. This enzyme is the focus of substantial attention, both because its structure and mechanism of action are still poorly studied, and because of its pivotal roles in aging and cellular proliferation. The use of telomerase as a potential target for the design of new anticancer drugs is also of great interest. The catalytic protein subunit of telomerase (TERT) contains an N-terminal domain (TEN) that is essential for activity and processivity. Elucidation of the structure and dynamics of TEN in solution is important for understanding the molecular mechanism of telomerase activity and for the design of new telomerase inhibitors. To approach this problem, in this study we report the (1)H, (13)C, and (15)N chemical shift assignments of TEN from Ogataea polymorpha. Analysis of the assigned chemical shifts allowed us to identify secondary structures and protein regions potentially involved in interaction with other participants of the telomerase catalytic cycle.

Novel multi-targeting anthra[2,3-b]thiophene-5,10-diones with guanidine-containing side chains: interaction with telomeric G-quadruplex, inhibition of telomerase and topoisomerase I and cytotoxic properties.

Novel generations of antitumor anthraquinones are expected to be advantageous over the conventional chemotherapeutic agents. Previous structure-activity relationship studies demonstrated an importance of the positively charged side chains conjugated to anthra[2,3-b]thiophene-5,10-dione scaffolds. Exploring a role of individual side chain moieties in binding to the duplex and G-quadruplex DNA, modulation of telomerase and topoisomerase I activities, intracellular accumulation and cytostatic potency, we herein analyzed a series of reported and newly synthesized guanidine-containing derivatives of anthra[2,3-b]thiophene-5,10-dione. We found that the number of cationic side chains (namely, two) is critical for a tight interaction with human telomeric G-quadruplex (TelQ). Along with a larger drug-TelQ association constant, the telomerase attenuation by anthrathiophenediones with two basic groups in the side chains was more pronounced than by the analogs bearing one basic group. For mono-guanidinated compounds the substituent with the amino group in the side chain provided better TelQ affinity than the methylamine residue. The intracellular uptake of the mono-guanidino derivative with two side chains was >2-fold higher than the respective value for the bis(guanidino) derivative. This difference can explain a lower antiproliferative potency of bis(guanidine) containing compounds. Thus, the modifications of side chains of anthra[2,3-b]thiophene-5,10-dione differently modulated drug-target interactions and cellular effects. Nevertheless, the selected compound 11-(3-aminopropylamino)-4-(2-guanidinoethylamino)anthra[2,3-b]thiophene-5,10-dione 13 demonstrated a high affinity to TelQ and the ability to stabilize the quadruplex structure. These properties were paralleled by reasonable potency of 13 as a telomerase/topoisomerase I inhibitor and an antiproliferative agent. These results indicate that the structural elements of anthra[2,3-b]thiophene-5,10-dione derivatives can be balanced to yield a candidate for further preclinical study.