Homologous recombination (HR) factors are crucial for DSB repair and processing stalled replication forks. RAD51 paralogs, including RAD51B, RAD51C, RAD51D, XRCC2 and XRCC3, have emerged as essential tumour suppressors, forming two subcomplexes, BCDX2 and CX3. Mutations in these genes are associated with cancer susceptibility and Fanconi anaemia, yet their biochemical activities remain unclear. This study reveals a linear arrangement of BCDX2 subunits compared to the RAD51 ring. BCDX2 shows a strong affinity towards single-stranded DNA (ssDNA) via unique binding mechanism compared to RAD51, and a contribution of DX2 subunits in binding branched DNA substrates. We demonstrate that BCDX2 facilitates RAD51 loading on ssDNA by suppressing the cooperative requirement of RAD51 binding to DNA and stabilizing the filament. Notably, BCDX2 also promotes RAD51 loading on short ssDNA and reversed replication fork substrates. Moreover, while mutants defective in ssDNA binding retain the ability to bind branched DNA substrates, they still facilitate RAD51 loading onto reversed replication forks. Our study provides mechanistic insights into how the BCDX2 complex stimulates the formation of BRCA2-independent RAD51 filaments on short stretches of ssDNA present at ssDNA gaps or stalled replication forks, highlighting its role in genome maintenance and DNA repair.
- MeSH
- DNA vazebné proteiny * metabolismus genetika MeSH
- jednovláknová DNA * metabolismus genetika MeSH
- lidé MeSH
- multiproteinové komplexy MeSH
- mutace MeSH
- rekombinasa Rad51 * metabolismus genetika MeSH
- replikace DNA * genetika MeSH
- vazba proteinů MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- časopisecké články MeSH
The recent human Monkeypox outbreak underlined the importance of studying basic biology of orthopoxviruses. However, the transcriptome of its causative agent has not been investigated before neither with short-, nor with long-read sequencing approaches. This Oxford Nanopore long-read RNA-Sequencing dataset fills this gap. It will enable the in-depth characterization of the transcriptomic architecture of the monkeypox virus, and may even make possible to annotate novel host transcripts. Moreover, our direct cDNA and native RNA sequencing reads will allow the estimation of gene expression changes of both the virus and the host cells during the infection. Overall, our study will lead to a deeper understanding of the alterations caused by the viral infection on a transcriptome level.
- MeSH
- komplementární DNA MeSH
- lidé MeSH
- nanopórové sekvenování * MeSH
- opičí neštovice * MeSH
- stanovení celkové genové exprese MeSH
- transkriptom MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- časopisecké články MeSH
- dataset MeSH
CONTEXT: Testing for BCR-ABL1 kinase domain (KD) mutations should always be performed before tyrosine kinase inhibitor (TKI) changes. Next-generation sequencing (NGS) is the best approach to highlight emerging mutations in patients not responding adequately to TKI therapy. However, NGS requires sample centralization and batch analysis and has a non-negligible time to results. In this study, we set up and validated a novel droplet digital PCR (ddPCR)-based multiplex strategy for the detection and quantitation of transcripts harboring mutations impacting TKI selection. METHODS: In collaboration with Bio-Rad, a 3-tube ddPCR strategy was designed that enables identification and quantitation of 16 nucleotide substitutions encoding the 13 mutations associated with resistance to one or more second-generation TKI (2GTKI). Primers and FAM-or FAM/HEX-labelled probes were grouped on a TKI-specific basis and generated clusters of droplets mapping to spatially distinct areas of the 2D plot based on resistance profiles. Each tube also incorporated primers and HEX-labelled probes for e13a2, e14a2, and e1a2 BCR::ABL1 fusion transcripts to express results as the percentage of mutation-positive over total BCR::ABL1 transcripts. For validation, a total of 101 RNA samples from healthy donors, TKI-sensitive and -resistant patients, and BCR::ABL1-positive and -negative cell lines were used. cDNA (125 ng) obtained with ABL1-specific primers was analyzed in duplicate on a QX200 ddPCR system (Bio-Rad). RESULTS: The limit of blank was determined using 60 blank samples. Accuracy and specificity were confirmed using 48 samples positive for one or more 2GTKI-resistant mutations or mutations at nearby codons (to exclude cross-reactivity). Analysis of serial dilutions of cell line mixtures made using BCR::ABL1-positive mutation-positive cells, BCR::ABL1-positive unmutated cells, and BCR::ABL1-negative cells to mimic different mutation frequencies (70%, 5%, and 0.5%) and different transcript levels (MR0 to MR3) showed that a 0.5% lower detection limit could be consistently achieved irrespective of BCR::ABL1 levels. CONCLUSIONS: ddPCR proved highly sensitive and accurate. Total hands-on time was approximately 2 hrs, and time from sample to results was 2 days. Therefore, ddPCR may be integrated into diagnostic algorithms of CML (and Ph+ ALL) patients as a convenient first-level screening tool for mutations impacting TKI selection.
- MeSH
- bcr-abl fúzní proteiny * genetika metabolismus MeSH
- chemorezistence genetika MeSH
- chronická myeloidní leukemie * diagnóza farmakoterapie genetika MeSH
- inhibitory proteinkinas farmakologie terapeutické užití MeSH
- komplementární DNA farmakologie MeSH
- lidé MeSH
- mutace MeSH
- nukleotidy MeSH
- polymerázová řetězová reakce MeSH
- RNA MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- časopisecké články MeSH
DNA synthesis of the leading and lagging strands works independently and cells tolerate single-stranded DNA generated during strand uncoupling if it is protected by RPA molecules. Natural alkaloid emetine is used as a specific inhibitor of lagging strand synthesis, uncoupling leading and lagging strand replication. Here, by analysis of lagging strand synthesis inhibitors, we show that despite emetine completely inhibiting DNA replication: it does not induce the generation of single-stranded DNA and chromatin-bound RPA32 (CB-RPA32). In line with this, emetine does not activate the replication checkpoint nor DNA damage response. Emetine is also an inhibitor of proteosynthesis and ongoing proteosynthesis is essential for the accurate replication of DNA. Mechanistically, we demonstrate that the acute block of proteosynthesis by emetine temporally precedes its effects on DNA replication. Thus, our results are consistent with the hypothesis that emetine affects DNA replication by proteosynthesis inhibition. Emetine and mild POLA1 inhibition prevent S-phase poly(ADP-ribosyl)ation. Collectively, our study reveals that emetine is not a specific lagging strand synthesis inhibitor with implications for its use in molecular biology.
The helicase domain of nonstructural protein 3 (NS3H) unwinds the double-stranded RNA replication intermediate in an ATP-dependent manner during the flavivirus life cycle. While the ATP hydrolysis mechanism of Dengue and Zika viruses NS3H has been extensively studied, little is known in the case of the tick-borne encephalitis virus NS3H. We demonstrate that ssRNA binds with nanomolar affinity to NS3H and strongly stimulates the ATP hydrolysis cycle, whereas ssDNA binds only weakly and inhibits ATPase activity in a noncompetitive manner. Thus, NS3H is an RNA-specific helicase, whereas DNA might act as an allosteric inhibitor. Using modeling, we explored plausible allosteric mechanisms by which ssDNA inhibits the ATPase via nonspecific binding in the vicinity of the active site and ATP repositioning. We captured several structural snapshots of key ATP hydrolysis stages using X-ray crystallography. One intermediate, in which the inorganic phosphate and ADP remained trapped inside the ATPase site after hydrolysis, suggests that inorganic phosphate release is the rate-limiting step. Using structure-guided modeling and molecular dynamics simulation, we identified putative RNA-binding residues and observed that the opening and closing of the ATP-binding site modulates RNA affinity. Site-directed mutagenesis of the conserved RNA-binding residues revealed that the allosteric activation of ATPase activity is primarily communicated via an arginine residue in domain 1. In summary, we characterized conformational changes associated with modulating RNA affinity and mapped allosteric communication between RNA-binding groove and ATPase site of tick-borne encephalitis virus helicase.
- MeSH
- adenosintrifosfát metabolismus MeSH
- adenosintrifosfatasy * metabolismus MeSH
- dvouvláknová RNA metabolismus MeSH
- fosfáty metabolismus MeSH
- jednovláknová DNA * metabolismus MeSH
- lidé MeSH
- RNA-helikasy * metabolismus MeSH
- virové nestrukturální proteiny * metabolismus MeSH
- viry klíšťové encefalitidy * enzymologie metabolismus MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
Isolation of RNA from whole saliva, a non-invasive and easily accessible biofluid that is an attractive alternative to blood for high-throughput biodosimetry of radiological/nuclear victims might be of clinical significance for prediction and diagnosis of disease. In a previous analysis of 12 human samples we identified two challenges to measuring gene expression from total RNA: (1) the fraction of human RNA in whole saliva was low and (2) the bacterial contamination was overwhelming. To overcome these challenges, we performed selective cDNA synthesis for human RNA species only by employing poly(A)+-tail primers followed by qRT-PCR. In the current study, this approach was independently validated on 91 samples from 61 healthy donors. Additionally, we used the ratio of human to bacterial RNA to adjust the input RNA to include equal amounts of human RNA across all samples before cDNA synthesis, which then ensured comparable analysis using the same base human input material. Furthermore, we examined relative levels of ten known housekeeping genes, and assessed inter- and intra-individual differences in 61 salivary RNA isolates, while considering effects of demographical factors (e.g. sex, age), epidemiological factors comprising social habits (e.g. alcohol, cigarette consumption), oral hygiene (e.g. flossing, mouthwash), previous radiological diagnostic procedures (e.g. number of CT-scans) and saliva collection time (circadian periodic). Total human RNA amounts appeared significantly associated with age only (P ≤ 0.02). None of the chosen housekeeping genes showed significant circadian periodicity and either did not associate or were weakly associated with the 24 confounders examined, with one exception, 60% of genes were altered by mouthwash. ATP6, ACTB and B2M represented genes with the highest mean baseline expression (Ct-values ≤ 30) and were detected in all samples. Combining these housekeeping genes for normalization purposes did not decrease inter-individual variance, but increased the robustness. In summary, our work addresses critical confounders and provides important information for the successful examination of gene expression in human whole saliva.
- MeSH
- bakteriální RNA MeSH
- dospělí MeSH
- esenciální geny * MeSH
- exprese genu * MeSH
- komplementární DNA MeSH
- kontaminace DNA MeSH
- kvantitativní polymerázová řetězová reakce MeSH
- lidé středního věku MeSH
- lidé MeSH
- mladý dospělý MeSH
- RNA izolace a purifikace MeSH
- sliny metabolismus MeSH
- stanovení celkové genové exprese metody MeSH
- Check Tag
- dospělí MeSH
- lidé středního věku MeSH
- lidé MeSH
- mladý dospělý MeSH
- mužské pohlaví MeSH
- ženské pohlaví MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
Covalent DNA interstrand crosslinks are toxic DNA damage lesions that block the replication machinery that can cause a genomic instability. Ubiquitous abasic DNA sites are particularly susceptible to spontaneous cross-linking with a base from the opposite DNA strand. Detection of a crosslink induces the DNA helicase ubiquitination that recruits NEIL3, a DNA glycosylase responsible for the lesion removal. NEIL3 utilizes several zinc finger domains indispensable for its catalytic NEI domain repairing activity. They recruit NEIL3 to the repair site and bind the single-stranded DNA. However, the molecular mechanism underlying their roles in the repair process is unknown. Here, we report the structure of the tandem zinc-finger GRF domain of NEIL3 and reveal the molecular details of its interaction with DNA. Our biochemical data indicate the preferential binding of the GRF domain to the replication fork. In addition, we obtained a structure for the catalytic NEI domain in complex with the DNA reaction intermediate that allowed us to construct and validate a model for the interplay between the NEI and GRF domains in the recognition of an interstrand cross-link. Our results suggest a mechanism for recognition of the DNA replication X-structure by NEIL3, a key step in the interstrand cross-link repair.
Metal-organic frameworks (MOFs) have been widely used as porous nanomaterials for different applications ranging from industrial to biomedicals. An unpredictable one-pot method is introduced to synthesize NH2-MIL-53 assisted by high-gravity in a greener media for the first time. Then, porphyrins were deployed to adorn the surface of MOF to increase the sensitivity of the prepared nanocomposite to the genetic materials and in-situ cellular protein structures. The hydrogen bond formation between genetic domains and the porphyrin' nitrogen as well as the surface hydroxyl groups is equally probable and could be considered a milestone in chemical physics and physical chemistry for biomedical applications. In this context, the role of incorporating different forms of porphyrins, their relationship with the final surface morphology, and their drug/gene loading efficiency were investigated to provide a predictable pattern in regard to the previous works. The conceptual phenomenon was optimized to increase the interactions between the biomolecules and the substrate by reaching the limit of detection to 10 pM for the Anti-cas9 protein, 20 pM for the single-stranded DNA (ssDNA), below 10 pM for the single guide RNA (sgRNA) and also around 10 nM for recombinant SARS-CoV-2 spike antigen. Also, the MTT assay showed acceptable relative cell viability of more than 85% in most cases, even by increasing the dose of the prepared nanostructures.
- MeSH
- buňky Hep G2 MeSH
- buňky PC12 MeSH
- COVID-19 diagnóza MeSH
- CRISPR-Cas systémy MeSH
- dusík chemie MeSH
- guide RNA, Kinetoplastida MeSH
- HEK293 buňky MeSH
- HeLa buňky MeSH
- jednovláknová DNA MeSH
- krysa rodu rattus MeSH
- lidé MeSH
- limita detekce MeSH
- nanokompozity MeSH
- nanostruktury MeSH
- porézní koordinační polymery chemie MeSH
- poréznost MeSH
- porfyriny chemie MeSH
- povrchové vlastnosti MeSH
- RNA virová metabolismus MeSH
- SARS-CoV-2 MeSH
- senzitivita a specificita MeSH
- testování na COVID-19 MeSH
- vodíková vazba MeSH
- zvířata MeSH
- Check Tag
- krysa rodu rattus MeSH
- lidé MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
The RAD51 recombinase assembles as helical nucleoprotein filaments on single-stranded DNA (ssDNA) and mediates invasion and strand exchange with homologous duplex DNA (dsDNA) during homologous recombination (HR), as well as protection and restart of stalled replication forks. Strand invasion by RAD51-ssDNA complexes depends on ATP binding. However, RAD51 can bind ssDNA in non-productive ADP-bound or nucleotide-free states, and ATP-RAD51-ssDNA complexes hydrolyse ATP over time. Here, we define unappreciated mechanisms by which the RAD51 paralog complex RFS-1/RIP-1 limits the accumulation of RAD-51-ssDNA complexes with unfavorable nucleotide content. We find RAD51 paralogs promote the turnover of ADP-bound RAD-51 from ssDNA, in striking contrast to their ability to stabilize productive ATP-bound RAD-51 nucleoprotein filaments. In addition, RFS-1/RIP-1 inhibits binding of nucleotide-free RAD-51 to ssDNA. We propose that 'nucleotide proofreading' activities of RAD51 paralogs co-operate to ensure the enrichment of active, ATP-bound RAD-51 filaments on ssDNA to promote HR.
- MeSH
- adenosindifosfát farmakologie MeSH
- adenosintrifosfát farmakologie MeSH
- Caenorhabditis elegans metabolismus MeSH
- druhová specificita MeSH
- fluorescence MeSH
- interferometrie MeSH
- jednovláknová DNA metabolismus MeSH
- nukleotidy metabolismus MeSH
- proteiny Caenorhabditis elegans metabolismus MeSH
- rekombinasa Rad51 chemie metabolismus MeSH
- sekvenční homologie aminokyselin * MeSH
- stabilita proteinů účinky léků MeSH
- vazba proteinů účinky léků MeSH
- zvířata MeSH
- Check Tag
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
RECQ5 is one of five RecQ helicases found in humans and is thought to participate in homologous DNA recombination by acting as a negative regulator of the recombinase protein RAD51. Here, we use kinetic and single molecule imaging methods to monitor RECQ5 behavior on various nucleoprotein complexes. Our data demonstrate that RECQ5 can act as an ATP-dependent single-stranded DNA (ssDNA) motor protein and can translocate on ssDNA that is bound by replication protein A (RPA). RECQ5 can also translocate on RAD51-coated ssDNA and readily dismantles RAD51-ssDNA filaments. RECQ5 interacts with RAD51 through protein-protein contacts, and disruption of this interface through a RECQ5-F666A mutation reduces translocation velocity by ∼50%. However, RECQ5 readily removes the ATP hydrolysis-deficient mutant RAD51-K133R from ssDNA, suggesting that filament disruption is not coupled to the RAD51 ATP hydrolysis cycle. RECQ5 also readily removes RAD51-I287T, a RAD51 mutant with enhanced ssDNA-binding activity, from ssDNA. Surprisingly, RECQ5 can bind to double-stranded DNA (dsDNA), but it is unable to translocate. Similarly, RECQ5 cannot dismantle RAD51-bound heteroduplex joint molecules. Our results suggest that the roles of RECQ5 in genome maintenance may be regulated in part at the level of substrate specificity.
- MeSH
- adenosintrifosfát metabolismus MeSH
- bodová mutace MeSH
- helikasy RecQ genetika metabolismus ultrastruktura MeSH
- homologní rekombinace * MeSH
- hydrolýza MeSH
- jednovláknová DNA metabolismus ultrastruktura MeSH
- kinetika MeSH
- lidé MeSH
- mikroskopie atomárních sil MeSH
- missense mutace MeSH
- molekulární motory metabolismus ultrastruktura MeSH
- rekombinantní fúzní proteiny metabolismus MeSH
- rekombinantní proteiny metabolismus MeSH
- rekombinasa Rad51 genetika metabolismus MeSH
- replikační protein A metabolismus MeSH
- substrátová specifita MeSH
- zobrazení jednotlivé molekuly * MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
- Research Support, N.I.H., Extramural MeSH
