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1.
Nat Methods ; 19(8): 959-968, 2022 08.
Artigo em Inglês | MEDLINE | ID: mdl-35927480

RESUMO

DNA-protein interactions mediate physiologic gene regulation and may be altered by DNA variants linked to polygenic disease. To enhance the speed and signal-to-noise ratio (SNR) in the identification and quantification of proteins associated with specific DNA sequences in living cells, we developed proximal biotinylation by episomal recruitment (PROBER). PROBER uses high-copy episomes to amplify SNR, and proximity proteomics (BioID) to identify the transcription factors and additional gene regulators associated with short DNA sequences of interest. PROBER quantified both constitutive and inducible association of transcription factors and corresponding chromatin regulators to target DNA sequences and binding quantitative trait loci due to single-nucleotide variants. PROBER identified alterations in regulator associations due to cancer hotspot mutations in the hTERT promoter, indicating that these mutations increase promoter association with specific gene activators. PROBER provides an approach to rapidly identify proteins associated with specific DNA sequences and their variants in living cells.


Assuntos
Cromatina , DNA , Biotinilação , Cromatina/genética , DNA/genética , DNA/metabolismo , Plasmídeos , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo
2.
Elife ; 112022 Aug 08.
Artigo em Inglês | MEDLINE | ID: mdl-35939393

RESUMO

The DNA sliding clamp proliferating cell nuclear antigen (PCNA) is an essential co-factor for many eukaryotic DNA metabolic enzymes. PCNA is loaded around DNA by the ATP-dependent clamp loader replication factor C (RFC), which acts at single-stranded (ss)/double-stranded DNA (dsDNA) junctions harboring a recessed 3' end (3' ss/dsDNA junctions) and at DNA nicks. To illuminate the loading mechanism we have investigated the structure of RFC:PCNA bound to ATPγS and 3' ss/dsDNA junctions or nicked DNA using cryogenic electron microscopy. Unexpectedly, we observe open and closed PCNA conformations in the RFC:PCNA:DNA complex, revealing that PCNA can adopt an open, planar conformation that allows direct insertion of dsDNA, and raising the question of whether PCNA ring closure is mechanistically coupled to ATP hydrolysis. By resolving multiple DNA-bound states of RFC:PCNA we observe that partial melting facilitates lateral insertion into the central channel formed by RFC:PCNA. We also resolve the Rfc1 N-terminal domain and demonstrate that its single BRCT domain participates in coordinating DNA prior to insertion into the central RFC channel, which promotes PCNA loading on the lagging strand of replication forks in vitro. Combined, our data suggest a comprehensive and fundamentally revised model for the RFC-catalyzed loading of PCNA onto DNA.


Assuntos
DNA , Proteínas de Saccharomyces cerevisiae , Trifosfato de Adenosina/metabolismo , DNA/metabolismo , Replicação do DNA , Antígeno Nuclear de Célula em Proliferação/genética , Antígeno Nuclear de Célula em Proliferação/metabolismo , Conformação Proteica , Proteína de Replicação C/química , Proteína de Replicação C/genética , Proteína de Replicação C/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo
3.
PLoS One ; 17(8): e0272645, 2022.
Artigo em Inglês | MEDLINE | ID: mdl-35939452

RESUMO

The Rad9-Rad1-Hus1 checkpoint clamp activates the DNA damage response and promotes DNA repair. DNA loading on the central channel of the Rad9-Rad1-Hus1 complex is required to execute its biological functions. Because Rad9A has the highest DNA affinity among the three subunits, we determined the domains and functional residues of human Rad9A that are critical for DNA interaction. The N-terminal globular domain (residues 1-133) had 3.7-fold better DNA binding affinity than the C-terminal globular domain (residues 134-266) of Rad9A1-266. Rad9A1-266 binds DNA 16-, 60-, and 30-fold better than Rad9A1-133, Rad9A134-266, and Rad9A94-266, respectively, indicating that different regions cooperatively contribute to DNA binding. We show that basic residues including K11, K15, R22, K78, K220, and R223 are important for DNA binding. The reductions on DNA binding of Ala substituted mutants of these basic residues show synergistic effect and are dependent on their residential Rad9A deletion constructs. Interestingly, deletion of a loop (residues 160-163) of Rad9A94-266 weakens DNA binding activity by 4.1-fold as compared to wild-type (WT) Rad9A94-266. Cellular sensitivity to genotoxin of rad9A knockout cells is restored by expressing WT-Rad9Afull. However, rad9A knockout cells expressing Rad9A mutants defective in DNA binding are more sensitive to H2O2 as compared to cells expressing WT-Rad9Afull. Only the rad9A knockout cells expressing loop-deleted Rad9A mutant are more sensitive to hydroxyurea than cells expressing WT-Rad9A. In addition, Rad9A-DNA interaction is required for DNA damage signaling activation. Our results indicate that DNA association by Rad9A is critical for maintaining cell viability and checkpoint activation under stress.


Assuntos
Exonucleases , Peróxido de Hidrogênio , Proteínas de Ciclo Celular/metabolismo , DNA/metabolismo , Dano ao DNA , Reparo do DNA , Exonucleases/genética , Humanos
4.
Proc Natl Acad Sci U S A ; 119(33): e2208004119, 2022 Aug 16.
Artigo em Inglês | MEDLINE | ID: mdl-35939705

RESUMO

The cohesin complex is required for sister chromatid cohesion and genome compaction. Cohesin coiled coils (CCs) can fold at break sites near midpoints to bring head and hinge domains, located at opposite ends of coiled coils, into proximity. Whether ATPase activities in the head play a role in this conformational change is yet to be known. Here, we dissected functions of cohesin ATPase activities in cohesin dynamics in Schizosaccharomyces pombe. Isolation and characterization of cohesin ATPase temperature-sensitive (ts) mutants indicate that both ATPase domains are required for proper chromosome segregation. Unbiased screening of spontaneous suppressor mutations rescuing the temperature lethality of cohesin ATPase mutants identified several suppressor hotspots in cohesin that located outside of ATPase domains. Then, we performed comprehensive saturation mutagenesis targeted to these suppressor hotspots. Large numbers of the identified suppressor mutations indicated several different ways to compensate for the ATPase mutants: 1) Substitutions to amino acids with smaller side chains in coiled coils at break sites around midpoints may enable folding and extension of coiled coils more easily; 2) substitutions to arginine in the DNA binding region of the head may enhance DNA binding; or 3) substitutions to hydrophobic amino acids in coiled coils, connecting the head and interacting with other subunits, may alter conformation of coiled coils close to the head. These results reflect serial structural changes in cohesin driven by its ATPase activities potentially for packaging DNAs.


Assuntos
Proteínas Cromossômicas não Histona , Schizosaccharomyces , Adenosina Trifosfatases/metabolismo , Aminoácidos/metabolismo , Proteínas de Ciclo Celular/metabolismo , Proteínas Cromossômicas não Histona/metabolismo , DNA/metabolismo , Schizosaccharomyces/genética , Schizosaccharomyces/metabolismo
5.
Nat Commun ; 13(1): 4624, 2022 Aug 08.
Artigo em Inglês | MEDLINE | ID: mdl-35941106

RESUMO

Argonaute proteins are programmable nucleases that have defense and regulatory functions in both eukaryotes and prokaryotes. All known prokaryotic Argonautes (pAgos) characterized so far act on DNA targets. Here, we describe a new class of pAgos that uniquely use DNA guides to process RNA targets. The biochemical and structural analysis of Pseudooceanicola lipolyticus pAgo (PliAgo) reveals an unusual organization of the guide binding pocket that does not rely on divalent cations and the canonical set of contacts for 5'-end interactions. Unconventional interactions of PliAgo with the 5'-phosphate of guide DNA define its new position within pAgo and shift the site of target RNA cleavage in comparison with known Argonautes. The specificity for RNA over DNA is defined by ribonucleotide residues at the cleavage site. The analysed pAgos sense mismatches and modifications in the RNA target. The results broaden our understanding of prokaryotic defense systems and extend the spectrum of programmable nucleases with potential use in RNA technology.


Assuntos
Proteínas Argonauta , RNA , Proteínas Argonauta/metabolismo , Bactérias/genética , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , DNA/metabolismo , Endonucleases/metabolismo , Células Procarióticas/metabolismo , RNA/metabolismo , RNA Guia/genética , RNA Guia/metabolismo
6.
Methods Mol Biol ; 2516: 39-50, 2022.
Artigo em Inglês | MEDLINE | ID: mdl-35922620

RESUMO

DNA methylations are one of the most well-known epigenetic modifications along with histone modifications and noncoding RNAs. They are found at specific sites along the DNA in all domains of life, with 5-mC and 6-mA/4-mC being well-characterized in eukaryotes and bacteria respectively, and they have not only been described as contributing to the structure of the double helix itself but also as regulators of DNA-based processes such as replication, transcription, and recombination. Different methods have been developed to accurately identify and/or map methylated motifs to decipher the involvement of DNA methylations in regulatory networks that affect the cellular state.Although DNA methylations have been detected along archaeal genomes, their involvement as regulators of DNA-based processes remains the least known. To highlight the importance of DNA methylations in the control of key cellular mechanisms and their dynamics in archaea cells, we have used single-molecule real-time (SMRT) sequencing. This sequencing technology allows the identification and direct mapping of the methylated motifs along the genome of an organism. In this chapter, we present a step-by-step protocol for detecting DNA methylations in the hyperthermophilic crenarchaeon Sulfolobus acidocaldarius using SMRT sequencing. This protocol can easily be adapted to other prokaryotes.


Assuntos
Sulfolobus acidocaldarius , DNA/metabolismo , Metilação de DNA , Genoma Arqueal , Sulfolobus acidocaldarius/genética , Sulfolobus acidocaldarius/metabolismo
7.
Methods Mol Biol ; 2516: 103-112, 2022.
Artigo em Inglês | MEDLINE | ID: mdl-35922624

RESUMO

DNA-binding transcription factors (TFs) play a central role in the gene expression of all organisms, from viruses to humans, including bacteria and archaea. The role of these proteins is the fate of gene expression in the context of environmental challenges. Because thousands of genomes have been sequenced to date, predictions of the encoded proteins are validated through the use of bioinformatics tools to obtain the necessary experimental, posterior knowledge. In this chapter, we describe three approaches to identify TFs in protein sequences. The first approach integrates the results of sequence comparisons and PFAM assignments, using as reference a manually curated collection of TFs. The second approach considers the prediction of DNA-binding structures, such as the classical helix-turn-helix (HTH); and the third approach considers a deep learning model. We suggest that all approaches must be considered together to increase the possibility of identifying new TFs in bacterial and archaeal genomes.


Assuntos
Genoma Arqueal , Fatores de Transcrição , Archaea/metabolismo , Bactérias/metabolismo , DNA/metabolismo , Genoma Arqueal/genética , Humanos , Fatores de Transcrição/metabolismo
8.
Methods Enzymol ; 672: 299-315, 2022.
Artigo em Inglês | MEDLINE | ID: mdl-35934481

RESUMO

Single-molecule imaging studies using long linear DNA substrates have revealed unanticipated insights into the dynamics of multi-protein systems. The use of long DNA substrates allows for the study of protein-DNA interactions with observation of the movement and behavior of proteins over distances accessible by fluorescence microscopy. Generalized methods can be exploited to generate and optimize a variety of linear DNA substrates with plasmid DNA as a simple starting point using standard biochemical techniques. Here, we present protocols to produce high-quality plasmid-based 36-kb linear DNA substrates that support DNA replication by the Escherichia coli replisome and that contain chemical lesions at well-defined positions. These substrates can be used to visualize replisome-lesion encounters at the single-molecule level, providing mechanistic details of replisome stalling and dynamics occurring during replication rescue and restart.


Assuntos
Replicação do DNA , DNA Polimerase Dirigida por DNA , DNA/metabolismo , DNA Polimerase III , DNA Polimerase Dirigida por DNA/química , Escherichia coli/genética , Escherichia coli/metabolismo
9.
Methods Enzymol ; 672: 369-381, 2022.
Artigo em Inglês | MEDLINE | ID: mdl-35934484

RESUMO

DNA topoisomerases resolve topological stress by introducing transient single- or double-strand breaks into the DNA duplex. This reaction requires the covalent binding of topoisomerases to DNA while the topological stress is being released. This transient intermediate is known as topoisomerase-covalent complex and represents the target of many anti-cancer drugs. Here, we describe a protocol to quantitatively detect topoisomerase-covalent complexes in vivo, called RADAR (rapid approach to DNA adduct recovery). DNA and protein-DNA covalent complexes are rapidly isolated from cells through chaotropic extraction. After normalization, samples are loaded on a slot blot, and the covalent complexes are detected using specific topoisomerase antibodies. In addition to being fast and robust, this assay produces quantitative results. Consequently, the RADAR assay can be applied to investigate the topoisomerase-covalent complex biology, including the effect of specific topoisomerase inhibitors. Finally, the same assay can be more generally applied to study covalent complexes of other enzymes with DNA.


Assuntos
DNA Topoisomerases Tipo I , DNA , DNA/metabolismo , DNA Topoisomerases Tipo I/metabolismo
10.
BMC Genomics ; 23(1): 556, 2022 Aug 04.
Artigo em Inglês | MEDLINE | ID: mdl-35927609

RESUMO

BACKGROUND: There is a need to investigate mechanisms of phenotypic plasticity in marine invertebrates as negative effects of climate change, like ocean acidification, are experienced by coastal ecosystems. Environmentally-induced changes to the methylome may regulate gene expression, but methylome responses can be species- and tissue-specific. Tissue-specificity has implications for gonad tissue, as gonad-specific methylation patterns may be inherited by offspring. We used the Pacific oyster (Crassostrea gigas) - a model for understanding pH impacts on bivalve molecular physiology due to its genomic resources and importance in global aquaculture- to assess how low pH could impact the gonad methylome. Oysters were exposed to either low pH (7.31 ± 0.02) or ambient pH (7.82 ± 0.02) conditions for 7 weeks. Whole genome bisulfite sequencing was used to identify methylated regions in female oyster gonad samples. C- > T single nucleotide polymorphisms were identified and removed to ensure accurate methylation characterization. RESULTS: Analysis of gonad methylomes revealed a total of 1284 differentially methylated loci (DML) found primarily in genes, with several genes containing multiple DML. Gene ontologies for genes containing DML were involved in development and stress response, suggesting methylation may promote gonad growth homeostasis in low pH conditions. Additionally, several of these genes were associated with cytoskeletal structure regulation, metabolism, and protein ubiquitination - commonly-observed responses to ocean acidification. Comparison of these DML with other Crassostrea spp. exposed to ocean acidification demonstrates that similar pathways, but not identical genes, are impacted by methylation. CONCLUSIONS: Our work suggests DNA methylation may have a regulatory role in gonad and larval development, which would shape adult and offspring responses to low pH stress. Combined with existing molluscan methylome research, our work further supports the need for tissue- and species-specific studies to understand the potential regulatory role of DNA methylation.


Assuntos
Crassostrea , Metilação de DNA , Animais , Crassostrea/metabolismo , DNA/metabolismo , Ecossistema , Feminino , Homeostase , Concentração de Íons de Hidrogênio , Oceanos e Mares , Água do Mar/química
11.
Stem Cell Res Ther ; 13(1): 397, 2022 Aug 04.
Artigo em Inglês | MEDLINE | ID: mdl-35927735

RESUMO

BACKGROUND: Diabetes-related osteoporosis (DOP) is a chronic disease caused by the high glucose environment that induces a metabolic disorder of osteocytes and osteoblast-associated mesenchymal stem cells. The processes of bone defect repair and regeneration become extremely difficult with DOP. Adipose-derived stem cells (ASCs), as seed cells in bone tissue engineering technology, provide a promising therapeutic approach for bone regeneration in DOP patients. The osteogenic ability of ASCs is lower in a DOP model than that of control ASCs. DNA methylation, as a mechanism of epigenetic regulation, may be involved in DNA methylation of various genes, thereby participating in biological behaviors of various cells. Emerging evidence suggests that increased DNA methylation levels are associated with activation of Wnt/ß-catenin signaling pathway. The purpose of this study was to investigate the influence of the diabetic environment on the osteogenic potential of ASCs, to explore the role of DNA methylation on osteogenic differentiation of DOP-ASCs via Wnt/ß-catenin signaling pathway, and to improve the osteogenic differentiation ability of ASCs with DOP. METHODS: DOP-ASCs and control ASCs were isolated from DOP C57BL/6 and control mice, respectively. The multipotency of DOP-ASCs was confirmed by Alizarin Red-S, Oil Red-O, and Alcian blue staining. Real-time polymerase chain reaction (RT-PCR), immunofluorescence, and western blotting were used to analyze changes in markers of osteogenic differentiation, DNA methylation, and Wnt/ß-catenin signaling. Alizarin Red-S staining was also used to confirm changes in the osteogenic ability. DNMT small interfering RNA (siRNA), shRNA-Dnmt3a, and LVRNA-Dnmt3a were used to assess the role of Dnmt3a in osteogenic differentiation of control ASCs and DOP-ASCs. Micro-computed tomography, hematoxylin and eosin staining, and Masson staining were used to analyze changes in the osteogenic capability while downregulating Dnmt3a with lentivirus in DOP mice in vivo. RESULTS: The proliferative ability of DOP-ASCs was lower than that of control ASCs. DOP-ASCs showed a decrease in osteogenic differentiation capacity, lower Wnt/ß-catenin signaling pathway activity, and a higher level of Dnmt3a than control ASCs. When Dnmt3a was downregulated by siRNA and shRNA, osteogenic-related factors Runt-related transcription factor 2 and osteopontin, and activity of Wnt/ß-catenin signaling pathway were increased, which rescued the poor osteogenic potential of DOP-ASCs. When Dnmt3a was upregulated by LVRNA-Dnmt3a, the osteogenic ability was inhibited. The same results were obtained in vivo. CONCLUSIONS: Dnmt3a silencing rescues the negative effects of DOP on ASCs and provides a possible approach for bone tissue regeneration in patients with diabetic osteoporosis.


Assuntos
Diabetes Mellitus , Osteoporose , Animais , Diferenciação Celular/fisiologia , Células Cultivadas , DNA/metabolismo , DNA Metiltransferase 3A , Diabetes Mellitus/genética , Regulação para Baixo , Epigênese Genética , Camundongos , Camundongos Endogâmicos C57BL , Osteogênese , Osteoporose/tratamento farmacológico , Osteoporose/terapia , RNA Interferente Pequeno/metabolismo , Células-Tronco/metabolismo , Via de Sinalização Wnt/genética , Microtomografia por Raio-X , beta Catenina/genética , beta Catenina/metabolismo
12.
Nat Commun ; 13(1): 3860, 2022 Jul 05.
Artigo em Inglês | MEDLINE | ID: mdl-35790757

RESUMO

DNA ligase I (LIG1) catalyzes the ligation of the nick repair intermediate after gap filling by DNA polymerase (pol) ß during downstream steps of the base excision repair (BER) pathway. However, how LIG1 discriminates against the mutagenic 3'-mismatches incorporated by polß at atomic resolution remains undefined. Here, we determine the X-ray structures of LIG1/nick DNA complexes with G:T and A:C mismatches and uncover the ligase strategies that favor or deter the ligation of base substitution errors. Our structures reveal that the LIG1 active site can accommodate a G:T mismatch in the wobble conformation, where an adenylate (AMP) is transferred to the 5'-phosphate of a nick (DNA-AMP), while it stays in the LIG1-AMP intermediate during the initial step of the ligation reaction in the presence of an A:C mismatch at the 3'-strand. Moreover, we show mutagenic ligation and aberrant nick sealing of dG:T and dA:C mismatches, respectively. Finally, we demonstrate that AP-endonuclease 1 (APE1), as a compensatory proofreading enzyme, removes the mismatched bases and interacts with LIG1 at the final BER steps. Our overall findings provide the features of accurate versus mutagenic outcomes coordinated by a multiprotein complex including polß, LIG1, and APE1 to maintain efficient repair.


Assuntos
Reparo do DNA , Mutagênicos , Monofosfato de Adenosina , DNA/metabolismo , Mutagênese
13.
Commun Biol ; 5(1): 667, 2022 Jul 05.
Artigo em Inglês | MEDLINE | ID: mdl-35790849

RESUMO

Efficient, accurate molecular characterization of genetically modified (GM) organisms is challenging, especially for those transgenic events transferred with genes/elements of recipient species. Herein, we decipher the comprehensive molecular characterization of one novel GM rice event G281 which was transferred with native promoters and an RNA interference (RNAi) expression cassette using paired-end whole genome sequencing (PE-WGS) and modified TranSeq approach. Our results show that transgenes integrate at rice chromosome 3 locus 16,439,674 included a 36 bp deletion of rice genomic DNA, and the whole integration contains two copies of the complete transfer DNA (T-DNA) in a head-to-head arrangement. No unintended insertion or backbone sequence of the transformed plasmid is observed at the whole genome level. Molecular characterization of the G281 event will assist risk assessment and application for a commercial license. In addition, we speculate that our approach could be further used for identifying the transgene integration of cisgenesis/intragenesis crops since both ends of T-DNA in G281 rice were from native gene or elements which is similar with that of cisgenesis/intrasgenesis. Our results from the in silico mimicking cisgenesis event confirm that the mimic rice Gt1 gene insertion and its flanking sequences are successfully identified, demonstrating the applicability of PE-WGS for molecular characterization of cisgenesis/intragenesis crops.


Assuntos
Oryza , Produtos Agrícolas/genética , DNA/metabolismo , Oryza/genética , Oryza/metabolismo , Plantas Geneticamente Modificadas/genética , Plantas Geneticamente Modificadas/metabolismo , Transgenes , Sequenciamento Completo do Genoma
14.
Sci Rep ; 12(1): 10856, 2022 Jul 07.
Artigo em Inglês | MEDLINE | ID: mdl-35798759

RESUMO

Listeria monocytogenes is a pathogenic bacterium that produces flagella, the locomotory organelles, in a temperature-dependent manner. At 37 °C inside humans, L. monocytogenes employs MogR to repress the expression of flagellar proteins, thereby preventing the production of flagella. However, in the low-temperature environment outside of the host, the antirepressor GmaR inactivates MogR, allowing flagellar formation. Additionally, DegU is necessary for flagellar expression at low temperatures. DegU transcriptionally activates the expression of GmaR and flagellar proteins by binding the operator DNA in the fliN-gmaR promoter as a response regulator of a two-component regulatory system. To determine the DegU-mediated regulation mechanism, we performed structural and biochemical analyses on the recognition of operator DNA by DegU. The DegU-DNA interaction is primarily mediated by a C-terminal DNA-binding domain (DBD) and can be fortified by an N-terminal receiver domain (RD). The DegU DBD adopts a tetrahelical helix-turn-helix structure and assembles into a dimer. The DegU DBD dimer recognizes the operator DNA using a positive patch. Unexpectedly, unlike typical response regulators, DegU interacts with operator DNA in both unphosphorylated and phosphorylated states with similar binding affinities. Therefore, we conclude that DegU is a noncanonical response regulator that is constitutively active irrespective of phosphorylation.


Assuntos
Listeria monocytogenes , Proteínas de Bactérias/metabolismo , DNA/metabolismo , Flagelos/fisiologia , Regulação Bacteriana da Expressão Gênica , Listeria monocytogenes/metabolismo , Regiões Promotoras Genéticas
15.
Cells ; 11(13)2022 Jul 02.
Artigo em Inglês | MEDLINE | ID: mdl-35805183

RESUMO

The load of DNA double-strand breaks (DSBs) induced in the genome of higher eukaryotes by different doses of ionizing radiation (IR) is a key determinant of DSB repair pathway choice, with homologous recombination (HR) and ATR substantially gaining ground at doses below 0.5 Gy. Increased resection and HR engagement with decreasing DSB-load generate a conundrum in a classical non-homologous end-joining (c-NHEJ)-dominated cell and suggest a mechanism adaptively facilitating resection. We report that ablation of DNA-PKcs causes hyper-resection, implicating DNA-PK in the underpinning mechanism. However, hyper-resection in DNA-PKcs-deficient cells can also be an indirect consequence of their c-NHEJ defect. Here, we report that all tested DNA-PKcs mutants show hyper-resection, while mutants with defects in all other factors of c-NHEJ fail to do so. This result rules out the model of c-NHEJ versus HR competition and the passive shift from c-NHEJ to HR as the causes of the increased resection and suggests the integration of DNA-PKcs into resection regulation. We develop a model, compatible with the results of others, which integrates DNA-PKcs into resection regulation and HR for a subset of DSBs. For these DSBs, we propose that the kinase remains at the break site, rather than the commonly assumed autophosphorylation-mediated removal from DNA ends.


Assuntos
Quebras de DNA de Cadeia Dupla , Proteínas de Ligação a DNA , DNA/metabolismo , Reparo do DNA por Junção de Extremidades , Proteínas de Ligação a DNA/metabolismo , Fenótipo
16.
Nutrients ; 14(13)2022 Jun 28.
Artigo em Inglês | MEDLINE | ID: mdl-35807853

RESUMO

The vitamin D receptor (VDR) mediates 1,25-dihydroxyvitamin D3 pleiotropic biological actions through transcription regulation of target genes. The expression levels of this ligand-activated nuclear receptor are regulated by multiple mechanisms both at transcriptional and post-transcriptional levels. Vitamin D3 is the natural VDR activator, but other molecules and signaling pathways have also been reported to regulate VDR expression and activity. In this study, we identify valproic acid (VPA) and natural short-chain fatty acids (SCFAs) as novel transcriptional activators of the human VDR (hVDR) gene. We further report a comprehensive characterization of VPA/SCFA-responsive elements in the 5' regulatory region of the hVDR gene. Two alternative promoter DNA regions (of 2.4 and 3.8 kb), as well as subsequent deletion fragments, were cloned in pGL4-LUC reporter vector. Transfection of these constructs in HepG2 and human Upcyte hepatocytes followed by reporter assays demonstrated that a region of 107 bp (from -107 to -1) upstream of the transcription start site in exon 1a is responsible for most of the increase in transcriptional activity in response to VPA/SCFAs. This short DNA region is GC-rich, does not contain an apparent TATA box, and includes two bona fide binding sites for the transcription factor Sp1. Our results substantiate the hypothesis that VPA and SCFAs facilitate the activity of Sp1 on novel Sp1 responsive elements in the hVDR gene, thus promoting VDR upregulation and signaling. Elevated hepatic VDR levels have been associated with liver steatosis and, therefore, our results may have clinical relevance in epileptic pediatric patients on VPA therapy. Our results could also be suggestive of VDR upregulation by SCFAs produced by gut microbiota.


Assuntos
Receptores de Calcitriol , Ácido Valproico , Sítios de Ligação , Criança , DNA/genética , DNA/metabolismo , Humanos , Regiões Promotoras Genéticas , Receptores de Calcitriol/genética , Receptores de Calcitriol/metabolismo , Fator de Transcrição Sp1/genética , Fator de Transcrição Sp1/metabolismo , Ácido Valproico/farmacologia
17.
J Int Med Res ; 50(7): 3000605221105344, 2022 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-35808817

RESUMO

OBJECTIVE: Chromodomain helicase DNA-binding 5 (CHD5) acts as a tumor suppressor gene in some cancers. CHD5 expression levels may affect an individual's susceptibility to hepatocellular carcinoma (HCC). This study aimed to evaluate the methylation pattern of the CHD5 promoter region and the gene's corresponding mRNA expression in HCC patients compared with healthy individuals. METHODS: In this case-control study, CHD5 mRNA gene expression levels and DNA methylation patterns were analyzed in 81 HCC patients and 90 healthy individuals by quantitative reverse transcription polymerase chain reaction and methylation-specific polymerase chain reaction, respectively. RESULTS: The CHD5 gene was hypermethylated in 61.8% of the HCC patients and 54.4% of the controls, and this difference was statistically significant. The CHD5 mRNA expression levels were significantly lower in the HCC patient group. CONCLUSIONS: Hypermethylation of the CHD5 promoter region may significantly lower the expression of this gene, affecting the incidence and severity of HCC. The methylation status of CHD5 can also be further studied as a prognostic factor in HCC.


Assuntos
Carcinoma Hepatocelular , Metilação de DNA , Neoplasias Hepáticas , RNA Mensageiro , Carcinoma Hepatocelular/genética , Estudos de Casos e Controles , DNA/metabolismo , DNA Helicases/genética , DNA Helicases/metabolismo , Epigênese Genética , Regulação Neoplásica da Expressão Gênica , Humanos , Neoplasias Hepáticas/genética , Proteínas do Tecido Nervoso/genética , Regiões Promotoras Genéticas , RNA Mensageiro/genética , RNA Mensageiro/metabolismo
18.
Cell Transplant ; 31: 9636897221108705, 2022.
Artigo em Inglês | MEDLINE | ID: mdl-35808831

RESUMO

Cooling at 4°C is routinely used to lower metabolism and preserve cell and tissue integrity in laboratory and clinical settings, including organ transplantation. However, cooling and rewarming produce cell damage, attributed primarily to a burst of reactive oxygen species (ROS) upon rewarming. While DNA represents a highly vulnerable target of ROS, it is unknown whether cooling and/or rewarming produces DNA damage. Here, we show that cooling alone suffices to produce extensive DNA damage in cultured primary cells and cell lines, including double-strand breaks (DSBs), as shown by comet assay and pulsed-field gel electrophoresis. Cooling-induced DSB formation is time- and temperature-dependent and coincides with an excess production of ROS, rather than a decrease in ATP levels. Immunohistochemistry confirmed that DNA damage activates the DNA damage response marked by the formation of nuclear foci of proteins involved in DSB repair, γ-H2Ax, and 53BP1. Subsequent rewarming for 24 h fails to recover ATP levels and only marginally lowers DSB amounts and nuclear foci. Precluding ROS formation by dopamine and the hydroxychromanol, Sul-121, dose-dependently reduces DSBs. Finally, a standard clinical kidney transplant procedure, using cold static storage in UW preservation solution up to 24 h in porcine kidney, lowered ATP, increased ROS, and produced increasing amounts of DSBs with recruitment of 53BP1. Given that DNA repair is erroneous by nature, cooling-inflicted DNA damage may affect cell survival, proliferation, and genomic stability, significantly impacting cellular and organ function, with relevance in stem cell and transplantation procedures.


Assuntos
Dano ao DNA , Histonas , Trifosfato de Adenosina/metabolismo , Animais , DNA/metabolismo , Histonas/metabolismo , Estresse Oxidativo , Espécies Reativas de Oxigênio/metabolismo , Suínos
19.
Methods Mol Biol ; 2525: 267-279, 2022.
Artigo em Inglês | MEDLINE | ID: mdl-35836075

RESUMO

Global hypomethylation of genomic DNA is associated with genomic instability and carcinogenic processes. The loss of DNA methylation has been reported in several cancers; therefore, global methylation levels have been considered as biomarkers for cancer diagnosis. Bisulfite conversion analysis has been widely used as the gold standard method for quantification of DNA methylation levels. However, this method requires cumbersome and time-consuming steps. To quantify global DNA methylation levels in homogeneous solutions, we exemplify a sensing system based on bioluminescence resonance energy transfer (BRET) using methyl-CpG binding domain (MBD)-fused firefly luciferase (MBD-FLuc) and unmethyl-CpG binding domain (CXXC)-fused firefly luciferase (CXXC-FLuc). MBD-FLuc and CXXC-FLuc bind to methylated and unmethylated CpGs, respectively, in the genomic DNA to excite BOBO-3, an intercalating dye on genomic DNA. These BOBO-3 emission intensities depend on the methylated and unmethylated CpG content. The global DNA methylation levels can be quantified from the BOBO-3 emission intensities. Moreover, we introduce a multicolor BRET assay using MBD-FLuc and CXXC-fused Oplophorus luciferase (CXXC-OLuc) for the simultaneous quantification of methylated and unmethylated CpG content in genomic DNA. CXXC-OLuc excites the BOBO-1 DNA-intercalating dye depending on the unmethylated CpG content. Thus, the emission intensities of BOBO-1 and BOBO-3 excited by CXXC-OLuc and MBD-FLuc, respectively, can be simultaneously measured, thereby enabling the determination of global DNA methylation level in a single step. Here, we describe the detailed protocols for the expression of MBD-FLuc, CXXC-FLuc, and CXXC-OLuc in Escherichia coli and determine the global DNA methylation levels using these BRET assays.


Assuntos
Metilação de DNA , Proteínas de Ligação a DNA , Ilhas de CpG , DNA/genética , DNA/metabolismo , Proteínas de Ligação a DNA/metabolismo , Transferência de Energia , Luciferases/metabolismo , Luciferases de Vaga-Lume/genética
20.
Front Immunol ; 13: 898724, 2022.
Artigo em Inglês | MEDLINE | ID: mdl-35795661

RESUMO

The innate immune response is the first-line host defense against pathogens. Cytosolic nucleic acids, including both DNA and RNA, represent a special type of danger signal to initiate an innate immune response. Activation of cytosolic nucleic acid sensors is tightly controlled in order to achieve the high sensitivity needed to combat infection while simultaneously preventing false activation that leads to pathologic inflammatory diseases. In this review, we focus on post-translational modifications of key cytosolic nucleic acid sensors that can reversibly or irreversibly control these sensor functions. We will describe phosphorylation, ubiquitination, SUMOylation, neddylation, acetylation, methylation, succinylation, glutamylation, amidation, palmitoylation, and oxidation modifications events (including modified residues, modifying enzymes, and modification function). Together, these post-translational regulatory modifications on key cytosolic DNA/RNA sensing pathway members reveal a complicated yet elegantly controlled multilayer regulator network to govern innate immune activation.


Assuntos
Ácidos Nucleicos , DNA/metabolismo , Processamento de Proteína Pós-Traducional , RNA/metabolismo , Transdução de Sinais
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