non-B structure
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Approximately 13% of the human genome at certain motifs have the potential to form noncanonical (non-B) DNA structures (e.g., G-quadruplexes, cruciforms, and Z-DNA), which regulate many cellular processes but also affect the activity of polymerases and helicases. Because sequencing technologies use these enzymes, they might possess increased errors at non-B structures. To evaluate this, we analyzed error rates, read depth, and base quality of Illumina, Pacific Biosciences (PacBio) HiFi, and Oxford Nanopore Technologies (ONT) sequencing at non-B motifs. All technologies showed altered sequencing success for most non-B motif types, although this could be owing to several factors, including structure formation, biased GC content, and the presence of homopolymers. Single-nucleotide mismatch errors had low biases in HiFi and ONT for all non-B motif types but were increased for G-quadruplexes and Z-DNA in all three technologies. Deletion errors were increased for all non-B types but Z-DNA in Illumina and HiFi, as well as only for G-quadruplexes in ONT. Insertion errors for non-B motifs were highly, moderately, and slightly elevated in Illumina, HiFi, and ONT, respectively. Additionally, we developed a probabilistic approach to determine the number of false positives at non-B motifs depending on sample size and variant frequency, and applied it to publicly available data sets (1000 Genomes, Simons Genome Diversity Project, and gnomAD). We conclude that elevated sequencing errors at non-B DNA motifs should be considered in low-read-depth studies (single-cell, ancient DNA, and pooled-sample population sequencing) and in scoring rare variants. Combining technologies should maximize sequencing accuracy in future studies of non-B DNA.
- MeSH
- hepatitida A MeSH
- hepatitida B MeSH
- hepatitida C MeSH
- imunoenzymatické techniky MeSH
- vakcíny proti virové hepatitidě MeSH
- Geografické názvy
- Estonsko MeSH
The complete genome sequence of non-type strain Clostridium pasteurianum NRRL B-598 was introduced last year; it is an oxygen tolerant, spore-forming, mesophilic heterofermentative bacterium with high hydrogen production and acetone-butanol fermentation ability. The basic genome statistics have shown its similarity to C. beijerinckii rather than the C. pasteurianum species. Here, we present a comparative analysis of the strain with several other complete clostridial genome sequences. Besides a 16S rRNA gene sequence comparison, digital DNA-DNA hybridization (dDDH) and phylogenomic analysis confirmed an inaccuracy of the taxonomic status of strain Clostridium pasteurianum NRRL B-598. Therefore, we suggest its reclassification to be Clostridium beijerinckii NRRL B-598. This is a specific strain and is not identical to other C. beijerinckii strains. This misclassification explains its unexpected behavior, different from other C. pasteurianum strains; it also permits better understanding of the bacterium for a future genetic manipulation that might increase its biofuel production potential.
- MeSH
- Clostridium beijerinckii klasifikace genetika MeSH
- Clostridium genetika MeSH
- DNA bakterií genetika MeSH
- fylogeneze MeSH
- genom bakteriální MeSH
- ribozomální DNA genetika MeSH
- RNA ribozomální 16S genetika MeSH
- sekvenční analýza DNA metody MeSH
- Publikační typ
- časopisecké články MeSH
- srovnávací studie MeSH
BACKGROUND: The breast and ovarian cancer susceptibility gene BRCA1 encodes a multifunctional tumor suppressor protein BRCA1, which is involved in regulating cellular processes such as cell cycle, transcription, DNA repair, DNA damage response and chromatin remodeling. BRCA1 protein, located primarily in cell nuclei, interacts with multiple proteins and various DNA targets. It has been demonstrated that BRCA1 protein binds to damaged DNA and plays a role in the transcriptional regulation of downstream target genes. As a key protein in the repair of DNA double-strand breaks, the BRCA1-DNA binding properties, however, have not been reported in detail. RESULTS: In this study, we provided detailed analyses of BRCA1 protein (DNA-binding domain, amino acid residues 444-1057) binding to topologically constrained non-B DNA structures (e.g. cruciform, triplex and quadruplex). Using electrophoretic retardation assay, atomic force microscopy and DNA binding competition assay, we showed the greatest preference of the BRCA1 DNA-binding domain to cruciform structure, followed by DNA quadruplex, with the weakest affinity to double stranded B-DNA and single stranded DNA. While preference of the BRCA1 protein to cruciform structures has been reported previously, our observations demonstrated for the first time a preferential binding of the BRCA1 protein also to triplex and quadruplex DNAs, including its visualization by atomic force microscopy. CONCLUSIONS: Our discovery highlights a direct BRCA1 protein interaction with DNA. When compared to double stranded DNA, such a strong preference of the BRCA1 protein to cruciform and quadruplex structures suggests its importance in biology and may thus shed insight into the role of these interactions in cell regulation and maintenance.
- MeSH
- B-DNA chemie metabolismus MeSH
- konformace nukleové kyseliny MeSH
- lidé MeSH
- protein BRCA1 chemie metabolismus MeSH
- proteinové domény MeSH
- vazba proteinů MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- časopisecké články MeSH
The human zinc transporter ZnT8 provides the granules of pancreatic β-cells with zinc (II) ions for assembly of insulin hexamers for storage. Until recently, the structure and function of human ZnTs have been modelled on the basis of the 3D structures of bacterial zinc exporters, which form homodimers with each monomer having six transmembrane α-helices harbouring the zinc transport site and a cytosolic domain with an α,β structure and additional zinc-binding sites. However, there are important differences in function as the bacterial proteins export an excess of zinc ions from the bacterial cytoplasm, whereas ZnT8 exports zinc ions into subcellular vesicles when there is no apparent excess of cytosolic zinc ions. Indeed, recent structural investigations of human ZnT8 show differences in metal binding in the cytosolic domain when compared to the bacterial proteins. Two common variants, one with tryptophan (W) and the other with arginine (R) at position 325, have generated considerable interest as the R-variant is associated with a higher risk of developing type 2 diabetes. Since the mutation is at the apex of the cytosolic domain facing towards the cytosol, it is not clear how it can affect zinc transport through the transmembrane domain. We expressed the cytosolic domain of both variants of human ZnT8 and have begun structural and functional studies. We found that (i) the metal binding of the human protein is different from that of the bacterial proteins, (ii) the human protein has a C-terminal extension with three cysteine residues that bind a zinc(II) ion, and (iii) there are small differences in stability between the two variants. In this investigation, we employed nickel(II) ions as a probe for the spectroscopically silent Zn(II) ions and utilised colorimetric and fluorimetric indicators for Ni(II) ions to investigate metal binding. We established Ni(II) coordination to the C-terminal cysteines and found differences in metal affinity and coordination in the two ZnT8 variants. These structural differences are thought to be critical for the functional differences regarding the diabetes risk. Further insight into the assembly of the metal centres in the cytosolic domain was gained from potentiometric investigations of zinc binding to synthetic peptides corresponding to N-terminal and C-terminal sequences of ZnT8 bearing the metal-coordinating ligands. Our work suggests the involvement of the C-terminal cysteines, which are part of the cytosolic domain, in a metal chelation and/or acquisition mechanism and, as now supported by the high-resolution structural work, provides the first example of metal-thiolate coordination chemistry in zinc transporters.
- MeSH
- beta-buňky metabolismus MeSH
- diabetes mellitus 2. typu genetika patologie MeSH
- inzulin genetika metabolismus MeSH
- konformace proteinů, alfa-helix genetika MeSH
- lidé MeSH
- molekulární konformace MeSH
- nikl chemie MeSH
- proteinové domény genetika MeSH
- transportní proteiny chemie genetika ultrastruktura MeSH
- vztahy mezi strukturou a aktivitou * MeSH
- zinek chemie MeSH
- zinkový transportér 8 chemie genetika ultrastruktura MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- časopisecké články MeSH
Léčebné možnosti nemocných s relabujícím/refrakterním agresivním non‑hodgkinským lymfomem (NHL) jsou omezené. Kombinace rituximabu s režimy obsahujícími antracykliny zůstávají základem terapie. Nicméně opakované podávání antracyklinů je spojeno se značným rizikem kardiotoxicity. Pixantron je cytostatikum antracyklinového typu se zachovanou antitumorózní aktivitou a sníženou kardiotoxicitou, které má prokazatelnou účinnost v léčbě NHL. Na základě výsledků mezinárodní randomizované studie PIX 301 byla monoterapie pixantronem schválena Evropskou lékovou agenturou (EMA) pro léčbu dospělých pacientů s opakovaně relabujícím či refrakterním agresivním B‑buněčným NHL.
reatment options for recurrent/refractory aggressive non‑Hodgkin's lymphoma (NHL) are limited. Rituximab combined with anthracycline based regimens remain the mainstay of therapy. However, repeated administration of anthracyclines is associated with substantial risk of cardiotoxicity. Pixantrone is anthracycline‑like cytostatic with preserved antitumor activity and decreased cardiotoxicity with proven efficacy in the treatment of NHL. On the basis of results from an international randomized PIX 301 trial, pixantrone monotherapy was approved by the European Medicines Agency (EMA) for the treatment of adults with multiply relapsed or refractory aggressive B‑NHL.
- Klíčová slova
- studie PIX 301, pixantron,
- MeSH
- antracykliny škodlivé účinky terapeutické užití MeSH
- difúzní velkobuněčný B-lymfom * farmakoterapie MeSH
- inhibitory topoisomerasy II * farmakologie terapeutické užití MeSH
- isochinoliny * farmakologie terapeutické užití MeSH
- kardiotoxicita MeSH
- klinické zkoušky jako téma MeSH
- kvantitativní vztahy mezi strukturou a aktivitou MeSH
- lidé MeSH
- nehodgkinský lymfom farmakoterapie MeSH
- neúspěšná terapie MeSH
- randomizované kontrolované studie jako téma MeSH
- Check Tag
- lidé MeSH
DNA conformation may deviate from the classical B-form in ∼13% of the human genome. Non-B DNA regulates many cellular processes; however, its effects on DNA polymerization speed and accuracy have not been investigated genome-wide. Such an inquiry is critical for understanding neurological diseases and cancer genome instability. Here, we present the first simultaneous examination of DNA polymerization kinetics and errors in the human genome sequenced with Single-Molecule Real-Time (SMRT) technology. We show that polymerization speed differs between non-B and B-DNA: It decelerates at G-quadruplexes and fluctuates periodically at disease-causing tandem repeats. Analyzing polymerization kinetics profiles, we predict and validate experimentally non-B DNA formation for a novel motif. We demonstrate that several non-B motifs affect sequencing errors (e.g., G-quadruplexes increase error rates), and that sequencing errors are positively associated with polymerase slowdown. Finally, we show that highly divergent G4 motifs have pronounced polymerization slowdown and high sequencing error rates, suggesting similar mechanisms for sequencing errors and germline mutations.
- MeSH
- DNA chemie MeSH
- G-kvadruplexy MeSH
- genomika * metody normy MeSH
- kinetika MeSH
- konformace nukleové kyseliny * MeSH
- lidé MeSH
- mutace MeSH
- nukleotidové motivy MeSH
- replikace DNA MeSH
- reprodukovatelnost výsledků MeSH
- sekvenční analýza DNA * metody MeSH
- vysoce účinné nukleotidové sekvenování * metody normy MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
BACKGROUND: Polyploidization is considered one of the main mechanisms of plant genome evolution. The presence of multiple copies of the same gene reduces selection pressure and permits sub-functionalization and neo-functionalization leading to plant diversification, adaptation and speciation. In bread wheat, polyploidization and the prevalence of transposable elements resulted in massive gene duplication and movement. As a result, the number of genes which are non-collinear to genomes of related species seems markedly increased in wheat. RESULTS: We used new-generation sequencing (NGS) to generate sequence of a Mb-sized region from wheat chromosome arm 3DS. Sequence assembly of 24 BAC clones resulted in two scaffolds of 1,264,820 and 333,768 bases. The sequence was annotated and compared to the homoeologous region on wheat chromosome 3B and orthologous loci of Brachypodium distachyon and rice. Among 39 coding sequences in the 3DS scaffolds, 32 have a homoeolog on chromosome 3B. In contrast, only fifteen and fourteen orthologs were identified in the corresponding regions in rice and Brachypodium, respectively. Interestingly, five pseudogenes were identified among the non-collinear coding sequences at the 3B locus, while none was found at the 3DS locus. CONCLUSION: Direct comparison of two Mb-sized regions of the B and D genomes of bread wheat revealed similar rates of non-collinear gene insertion in both genomes with a majority of gene duplications occurring before their divergence. Relatively low proportion of pseudogenes was identified among non-collinear coding sequences. Our data suggest that the pseudogenes did not originate from insertion of non-functional copies, but were formed later during the evolution of hexaploid wheat. Some evidence was found for gene erosion along the B genome locus.
- MeSH
- Brachypodium genetika MeSH
- chromozomy rostlin genetika MeSH
- DNA rostlinná genetika MeSH
- duplikace genu MeSH
- fylogeneze MeSH
- genetické lokusy genetika MeSH
- genom rostlinný genetika MeSH
- inzerční mutageneze MeSH
- kontigové mapování MeSH
- molekulární evoluce * MeSH
- polyploidie MeSH
- pšenice genetika MeSH
- pseudogeny genetika MeSH
- rýže (rod) genetika MeSH
- sekvenční analýza DNA MeSH
- umělé bakteriální chromozomy MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
- srovnávací studie MeSH
