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Cell cycle progression is linked to transcriptome dynamics and variations in the response of pluripotent cells to differentiation cues, mostly through unknown determinants. Here, we characterized the cell-cycle-associated transcriptome and proteome of mouse embryonic stem cells (mESCs) in naive ground state. We found that the thymine DNA glycosylase (TDG) is a cell-cycle-regulated co-factor of the tumor suppressor p53. Furthermore, TDG and p53 co-bind ESC-specific cis-regulatory elements and thereby control transcription of p53-dependent genes during self-renewal. We determined that the dynamic expression of TDG is required to promote the cell-cycle-associated transcriptional heterogeneity. Moreover, we demonstrated that transient depletion of TDG influences cell fate decisions during the early differentiation of mESCs. Our findings reveal an unanticipated role of TDG in promoting molecular heterogeneity during the cell cycle and highlight the central role of protein dynamics for the temporal control of cell fate during development.
Assuntos
Timina DNA Glicosilase , Proteína Supressora de Tumor p53 , Animais , Camundongos , Ciclo Celular/genética , Linhagem Celular , Regulação da Expressão Gênica , Timina DNA Glicosilase/genética , Timina DNA Glicosilase/metabolismo , Proteína Supressora de Tumor p53/genética , Proteína Supressora de Tumor p53/metabolismoRESUMO
The DNA replication of mpox virus is performed by the viral polymerase F8 and also requires other viral factors, including processivity factor A22, uracil DNA glycosylase E4, and phosphoprotein H5. However, the molecular roles of these viral factors remain unclear. Here, we characterize the structures of F8-A22-E4 and F8-A22-E4-H5 complexes in the presence of different primer-template DNA substrates. E4 is located upstream of F8 on the template single-stranded DNA (ssDNA) and is catalytically active, highlighting a functional coupling between DNA base-excision repair and DNA synthesis. Moreover, H5, in the form of tetramer, binds to the double-stranded DNA (dsDNA) region downstream of F8 in a similar position as PCNA (proliferating cell nuclear antigen) does in eukaryotic polymerase complexes. Omission of H5 or disruption of its DNA interaction showed a reduced synthesis of full-length DNA products. These structures provide snapshots for the working cycle of the polymerase and generate insights into the mechanisms of these essential factors in viral DNA replication.
Assuntos
Replicação do DNA , DNA Polimerase Dirigida por DNA , DNA Polimerase Dirigida por DNA/metabolismo , Monkeypox virus/genética , Monkeypox virus/metabolismo , Replicação Viral , DNA Viral/genética , Antígeno Nuclear de Célula em Proliferação/genética , Antígeno Nuclear de Célula em Proliferação/metabolismoRESUMO
NEIL1 is a DNA glycosylase that recognizes and initiates base excision repair of oxidized bases. The ubiquitous ssDNA binding scaffolding protein, replication protein A (RPA), modulates NEIL1 activity in a manner that depends on DNA structure. Interaction between NEIL1 and RPA has been reported, but the molecular basis of this interaction has yet to be investigated. Using a combination of NMR spectroscopy and isothermal titration calorimetry (ITC), we show that NEIL1 interacts with RPA through two contact points. An interaction with the RPA32C protein recruitment domain was mapped to a motif in the common interaction domain (CID) of NEIL1 and a dissociation constant (Kd) of 200 nM was measured. A substantially weaker secondary interaction with the tandem RPA70AB ssDNA binding domains was also mapped to the CID. Together these two contact points reveal NEIL1 has a high overall affinity (Kd â¼ 20 nM) for RPA. A homology model of the complex of RPA32C with the NEIL1 RPA binding motif in the CID was generated and used to design a set of mutations in NEIL1 to disrupt the interaction, which was confirmed by ITC. The mutant NEIL1 remains catalytically active against a thymine glycol lesion in duplex DNA in vitro. Testing the functional effect of disrupting the NEIL1-RPA interaction in vivo using a Fluorescence Multiplex-Host Cell Reactivation (FM-HCR) reporter assay revealed an unexpected role for NEIL1 in nucleotide excision repair. These findings are discussed in the context of the role of NEIL1 in replication-associated repair.
RESUMO
Oxidative DNA damage-related diseases, such as incurable inflammation, malignant tumors, and age-related disorders, present significant challenges in modern medicine due to their complex molecular mechanisms and limitations in identifying effective treatment targets. Recently, 8-oxoguanine DNA glycosylase 1 (OGG1) has emerged as a promising multifunctional therapeutic target for the treatment of these challenging diseases. In this review, we systematically summarize the multiple functions and mechanisms of OGG1, including pro-inflammatory, tumorigenic, and aging regulatory mechanisms. We also highlight the potential of OGG1 inhibitors and activators as potent therapeutic agents for the aforementioned life-limiting diseases. We conclude that OGG1 serves as a multifunctional hub; the inhibition of OGG1 may provide a novel approach for preventing and treating inflammation and cancer, and the activation of OGG1 could be a strategy for preventing age-related disorders. Furthermore, we provide an extensive overview of successful applications of OGG1 regulation in treating inflammatory, cancerous, and aging-related diseases. Finally, we discuss the current challenges and future directions of OGG1 as an emerging multifunctional therapeutic marker for the aforementioned challenging diseases. The aim of this review is to provide a robust reference for scientific researchers and clinical drug developers in the development of novel clinical targeted drugs for life-limiting diseases, especially for incurable inflammation, malignant tumors, and age-related disorders.
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Thymine DNA glycosylase (TDG) is an essential enzyme involved in numerous biological pathways, including DNA repair, DNA demethylation, and transcriptional activation. Despite these important functions, the mechanisms surrounding the actions and regulation of TDG are poorly understood. In this study, we demonstrate that TDG induces phase separation of DNA and nucleosome arrays under physiologically relevant conditions in vitro and show that the resulting chromatin droplets exhibited behaviors typical of phase-separated liquids, supporting a liquid-liquid phase separation model. We also provide evidence that TDG has the capacity to form phase-separated condensates in the cell nucleus. The ability of TDG to induce chromatin phase separation is dependent on its intrinsically disordered N- and C-terminal domains, which in isolation, promote the formation of chromatin-containing droplets having distinct physical properties, consistent with their unique mechanistic roles in the phase separation process. Interestingly, DNA methylation alters the phase behavior of the disordered domains of TDG and compromises formation of chromatin condensates by full-length TDG, indicating that DNA methylation regulates the assembly and coalescence of TDG-mediated condensates. Overall, our results shed new light on the formation and physical nature of TDG-mediated chromatin condensates, which have broad implications for the mechanism and regulation of TDG and its associated genomic processes.
Assuntos
Cromatina , Metilação de DNA , DNA , Timina DNA Glicosilase , Cromatina/química , Cromatina/genética , Cromatina/metabolismo , DNA/química , DNA/metabolismo , Timina DNA Glicosilase/genética , Timina DNA Glicosilase/metabolismo , Nucleossomos/química , Nucleossomos/genética , Nucleossomos/metabolismoRESUMO
Thymine DNA glycosylase (TDG) is a multifaceted enzyme involved in several critical biological pathways, including transcriptional activation, DNA demethylation, and DNA repair. Recent studies have established regulatory relationships between TDG and RNA, but the molecular interactions underlying these relationships are poorly understood. Herein, we now demonstrate that TDG binds directly to RNA with nanomolar affinity. Using synthetic oligonucleotides of defined length and sequence, we show that TDG has a strong preference for binding G-rich sequences in single-stranded RNA but binds weakly to single-stranded DNA and duplex RNA. TDG also binds tightly to endogenous RNA sequences. Studies with truncated proteins indicate that TDG binds RNA primarily through its structured catalytic domain and that its disordered C-terminal domain plays a key role in regulating TDG's affinity and selectivity for RNA. Finally, we show that RNA competes with DNA for binding to TDG, resulting in the inhibition of TDG-mediated excision in the presence of RNA. Together, this work provides support for and insights into a mechanism wherein TDG-mediated processes (e.g., DNA demethylation) are regulated through the direct interactions of TDG with RNA.
Assuntos
Timina DNA Glicosilase , Timina DNA Glicosilase/genética , Timina DNA Glicosilase/metabolismo , Reparo do DNA , DNA/metabolismo , RNA , Proteínas de Ligação a RNA/metabolismo , TiminaRESUMO
Base editing enables precise gene editing without requiring donor DNA or double-stranded breaks. To facilitate base editing tools, a uracil DNA glycosylase inhibitor (UGI) was fused to cytidine deaminase-Cas nickase to inhibit uracil DNA glycosylase (UDG). Herein, we revealed that the bacteriophage PBS2-derived UGI of the cytosine base editor (CBE) could not inhibit archaic Type IV UDG in oligoploid cyanobacteria. To overcome the limitation of the CBE, dCas12a-assisted gene repression of the udg allowed base editing at the desired targets with up to 100% mutation frequencies, and yielded correct phenotypes of desired mutants in cyanobacteria. Compared with the original CBE (BE3), base editing was analyzed within a broader C4-C16 window with a strong TC-motif preference. Using multiplexed CyanoCBE, while udg was repressed, simultaneous base editing at two different sites was achieved with lower mutation frequencies than single CBE. Our discovery of a Type IV UDG that is not inhibited by the UGI of the CBE in cyanobacteria and the development of dCas12a-mediated base editing should facilitate the application of base editing not only in cyanobacteria, but also in archaea and green algae that possess Type IV UDGs. We revealed the bacteriophage-derived UGI of the base editor did not repress Type IV UDG in cyanobacteria. To overcome the limitation, orthogonal dCas12a interference was successfully applied to repress the UDG gene expression in cyanobacteria during base editing occurred, yielding a premature translational termination at desired targets. This study will open a new opportunity to perform base editing with Type IV UDGs in archaea and green algae.
Assuntos
Cianobactérias , Uracila-DNA Glicosidase , Uracila-DNA Glicosidase/genética , Uracila-DNA Glicosidase/metabolismo , Edição de Genes , DNA , Reparo do DNA , Cianobactérias/genética , Cianobactérias/metabolismo , CitosinaRESUMO
Repurposing drugs can significantly reduce the time and costs associated with drug discovery and development. However, many drug compounds possess intrinsic fluorescence, resulting in aberrations such as auto-fluorescence, scattering and quenching, in fluorescent high-throughput screening assays. To overcome these drawbacks, time-resolved technologies have received increasing attention. In this study, we have developed a rapid and efficient screening platform based on time-resolved emission spectroscopy in order to screen for inhibitors of the DNA repair enzyme, uracil-DNA glycosylase (UDG). From a database of 1456 FDA/EMA-approved drugs, sodium stibogluconate was discovered as a potent UDG inhibitor. This compound showed synergistic cytotoxicity against 5-fluorouracil-resistant cancer cells. This work provides a promising future for time-resolved technologies for high-throughput screening (HTS), allowing for the swift identification of bioactive compounds from previously overlooked scaffolds due to their inherent fluorescence properties.
Assuntos
Neoplasias da Próstata , Uracila-DNA Glicosidase , Humanos , Masculino , Uracila-DNA Glicosidase/química , Oligonucleotídeos , Gluconato de Antimônio e Sódio , Avaliação Pré-Clínica de Medicamentos , Reposicionamento de Medicamentos , Detecção Precoce de CâncerRESUMO
The generation of DNA damage causes mutations and consequently cancer. Reactive oxygen species are important sources of DNA damage and some mutation signatures found in human cancers. 8-Oxo-7,8-dihydroguanine (GO, 8-hydroxyguanine) is one of the most abundant oxidized bases and induces a GâT transversion mutation at the modified site. The damaged G base also causes untargeted base substitution mutations at the G bases of 5'-GpA-3' dinucleotides (action-at-a-distance mutations) in human cells, and the cytosine deaminase apolipoprotein B mRNA-editing enzyme, catalytic polypeptide-like 3 (APOBEC3) is involved in the mutation process. The deaminated cytosine, i.e., uracil, bases are expected to be removed by uracil DNA glycosylase. Most of the substitution mutations at the G bases of 5'-GpA-3' might be caused by abasic sites formed by the glycosylase. In this study, we expressed the uracil DNA glycosylase inhibitor from Bacillus subtilis bacteriophage PBS2 in human U2OS cells and examined the effects on the GO-induced action-at-a-distance mutations. The inhibition of uracil DNA glycosylase increased the mutation frequency, and in particular, the frequency of GâA transitions. These results indicated that uracil DNA glycosylase, in addition to APOBEC3, is involved in the untargeted mutation process induced by GO.
Assuntos
Guanina , Mutação , Uracila-DNA Glicosidase , Humanos , Guanina/análogos & derivados , Guanina/metabolismo , Uracila-DNA Glicosidase/metabolismo , Uracila-DNA Glicosidase/genética , Linhagem Celular Tumoral , Dano ao DNA , Bacillus subtilis/genética , Bacteriófagos/genéticaRESUMO
Hydroxyhydroquinone (HHQ) is an oxidative component produced by roasting coffee beans and has been reported to generate relatively large amounts of reactive oxygen species (ROS). In this study, we used senescence-accelerated mouse prone 8 (SAMP8) mice to determine whether HHQ consumption increases oxidative-stress-induced injury, because in SAMP8 mice, the activity of 8-oxoguanine DNA glycosylase 1, which repairs oxidative modifications in DNA, is decreased. The results showed that two out of twelve (16.7%) HHQ-treated mice presented polyuria and glucosuria around 2 months after the start of treatment, indicating that HHQ may act as a mutagen against SAMP8 mice, which is sensitive to oxidative damage. No abnormalities were observed in the chlorogenic acid (coffee polyphenol, CPP)-treated group. The concentration of hydrogen peroxide in the serum of SAMP8 mice was significantly higher than that in SAMR1 (senescence-resistant) control mice, and the concentration was further increased in the HHQ-treated group. CPP, when coexisting with HHQ at the rate contained in roasted coffee, decreased the amount of hydrogen peroxide in the serum of SAMP8 mice. Although CPP can act both oxidatively and antioxidatively as a polyphenol, CPP acts more antioxidatively when coexisting with HHQ. Thus, the oxidative effect of HHQ was shown to be counteracted by CPP.
Assuntos
Ácido Clorogênico , Hidroquinonas , Polifenóis , Animais , Camundongos , Ácido Clorogênico/farmacologia , Polifenóis/farmacologia , Mutagênicos/toxicidade , Peróxido de Hidrogênio , Estresse Oxidativo , DNARESUMO
The base excision repair (BER) pathway historically has been associated with maintaining genome integrity by eliminating nucleobases with small chemical modifications. In the past several years, however, BER was found to play additional roles in genome maintenance and metabolism, including sequence-specific restriction modification and repair of bulky adducts and interstrand crosslinks. Central to this expanded biological utility are specialized DNA glycosylases - enzymes that selectively excise damaged, modified, or mismatched nucleobases. In this review we discuss the newly identified roles of the BER pathway and examine the structural and mechanistic features of the DNA glycosylases that enable these functions.
Assuntos
DNA Glicosilases/metabolismo , Reparo do DNA , DNA/metabolismo , DNA/química , Dano ao DNA , DNA Glicosilases/química , HumanosRESUMO
Parkinson's disease (PD) is a common movement disorder caused by a characteristic loss of dopaminergic neurons in the substantia nigra and degeneration of dopamine terminals in the dorsal striatum. Previous studies have suggested that oxidative stress-induced DNA damage may be involved in PD pathogenesis, as steady-state levels of several types of oxidized nucleobases were shown to be elevated in PD brain tissues. These DNA lesions are normally removed from the genome by base excision repair, which is initiated by DNA glycosylase enzymes such as endonuclease VIII-like 1 (Neil1). In this study, we show that Neil1 plays an important role in limiting oxidative stress-induced degeneration of dopaminergic neurons. In particular, Neil1-deficient male mice exhibited enhanced sensitivity to nigrostriatal degeneration after 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) administration, and Neil1-deficient animals had higher levels of γH2AX-marked DNA damage than wild-type (WT) controls, regardless of treatment status. Moreover, MPTP-treated Neil1-/- male mice had slightly elevated expression of genes related to the nuclear factor erythroid 2-related factor 2 (Nrf2)-dependent antioxidant pathway. Treatment with the Nrf2 activator, monomethyl fumarate, reduced PD-like behaviors and pathology in Neil1-/- male mice, suggesting that Neil1 is an important defense molecule in an oxidative cellular environment. Taken together, these results suggest that Neil1 DNA glycosylase may play an important role in limiting oxidative stress-mediated PD pathogenesis.
Assuntos
DNA Glicosilases , Doença de Parkinson , Masculino , Camundongos , Animais , Doença de Parkinson/metabolismo , 1-Metil-4-Fenil-1,2,3,6-Tetra-Hidropiridina/metabolismo , 1-Metil-4-Fenil-1,2,3,6-Tetra-Hidropiridina/farmacologia , Desoxirribonuclease (Dímero de Pirimidina)/metabolismo , Neurônios Dopaminérgicos/metabolismo , Fator 2 Relacionado a NF-E2/genética , Fator 2 Relacionado a NF-E2/metabolismo , Substância Negra/patologia , DNA Glicosilases/genética , DNA Glicosilases/metabolismo , Camundongos Endogâmicos C57BL , Modelos Animais de Doenças , Corpo Estriado/metabolismoRESUMO
BACKGROUND: H2A.X is an H2A variant histone in eukaryotes, unique for its ability to respond to DNA damage, initiating the DNA repair pathway. H2A.X replacement within the histone octamer is mediated by the FAcilitates Chromatin Transactions (FACT) complex, a key chromatin remodeler. FACT is required for DEMETER (DME)-mediated DNA demethylation at certain loci in Arabidopsis thaliana female gametophytes during reproduction. Here, we sought to investigate whether H2A.X is involved in DME- and FACT-mediated DNA demethylation during reproduction. RESULTS: H2A.X is encoded by two genes in Arabidopsis genome, HTA3 and HTA5. We generated h2a.x double mutants, which displayed a normal growth profile, whereby flowering time, seed development, and root tip organization, S-phase progression and proliferation were all normal. However, h2a.x mutants were more sensitive to genotoxic stress, consistent with previous reports. H2A.X fused to Green Fluorescent Protein (GFP) under the H2A.X promoter was highly expressed especially in newly developing Arabidopsis tissues, including in male and female gametophytes, where DME is also expressed. We examined DNA methylation in h2a.x developing seeds and seedlings using whole genome bisulfite sequencing, and found that CG DNA methylation is decreased genome-wide in h2a.x mutant endosperm. Hypomethylation was most striking in transposon bodies, and occurred on both parental alleles in the developing endosperm, but not the embryo or seedling. h2a.x-mediated hypomethylated sites overlapped DME targets, but also included other loci, predominately located in heterochromatic transposons and intergenic DNA. CONCLUSIONS: Our genome-wide methylation analyses suggest that H2A.X could function in preventing access of the DME demethylase to non-canonical sites. Overall, our data suggest that H2A.X is required to maintain DNA methylation homeostasis in the unique chromatin environment of the Arabidopsis endosperm.
Assuntos
Proteínas de Arabidopsis , Arabidopsis , Arabidopsis/genética , Arabidopsis/metabolismo , Metilação de DNA/genética , Endosperma/genética , Endosperma/metabolismo , Histonas/genética , Histonas/metabolismo , Proteínas de Arabidopsis/genética , Proteínas de Arabidopsis/metabolismo , Cromatina , Regulação da Expressão Gênica de PlantasRESUMO
Alkylated bases in DNA created in the presence of endogenous and exogenous alkylating agents are either cytotoxic or mutagenic, or both to a cell. Currently, cells have evolved several strategies for repairing alkylated base. One strategy is a base excision repair process triggered by a specific DNA glycosylase that is used for the repair of the cytotoxic 3-methyladenine. Additionally, the cytotoxic and mutagenic O6-methylguanine (O6-meG) is corrected by O6-methylguanine methyltransferase (MGMT) via directly transferring the methyl group in the lesion to a specific cysteine in this protein. Furthermore, oxidative DNA demethylation catalyzed by DNA dioxygenase is utilized for repairing the cytotoxic 3-methylcytosine (3-meC) and 1-methyladenine (1-meA) in a direct reversal manner. As the third domain of life, Archaea possess 3-methyladenine DNA glycosylase II (AlkA) and MGMT, but no DNA dioxygenase homologue responsible for oxidative demethylation. Herein, we summarize recent progress in structural and biochemical properties of archaeal AlkA and MGMT to gain a better understanding of archaeal DNA alkylation repair, focusing on similarities and differences between the proteins from different archaeal species and between these archaeal proteins and their bacterial and eukaryotic relatives. To our knowledge, it is the first review on archaeal DNA alkylation repair conducted by DNA glycosylase and methyltransferase. KEY POINTS: ⢠Archaeal MGMT plays an essential role in the repair of O 6 -meG ⢠Archaeal AlkA can repair 3-meC and 1-meA.
Assuntos
DNA Glicosilases , Dioxigenases , Metiltransferases/genética , DNA Arqueal/genética , Alquilação , DNA Glicosilases/metabolismo , DNA/metabolismo , Dioxigenases/metabolismoRESUMO
If left unrepaired, the major oxidative DNA lesion 7,8-dihydro-8-oxoguanine (oxoG) promotes G-to-T transversions by favorably adopting a syn conformation and base pairing with dATP during replication. The human oxoG DNA glycosylase hOGG1 senses and removes oxoG amid millions-fold excess of guanine, thereby counteracting the genotoxic effects of the major oxidative damage. Crystal structures of hOGG1 in complex with oxoG-containing DNA have provided key insights into the lesion recognition and catalysis mechanisms of the enzyme. These lesion-recognition complex (LRC) structures typically involve a catalytically inactive hOGG1 mutant, where one of the catalytic-site amino acid residues is mutated to prevent the cleavage of oxoG. The use of a catalytically incompetent hOGG1 mutant has thus precluded understanding of unscathed interactions between oxoG and hOGG1 catalytic site as well as interactions among catalytic-site amino acid residues. As an orthogonal approach to visualize such interactions, we have co-crystallized a catalytically competent hOGG1 bound to 2'-fluoro-oxodG-containing DNA, a transition state destabilizing inhibitor that binds hOGG1 but is not processed by the enzyme. In this fluorinated lesion-recognition complex (FLRC), the 8-oxo moiety of oxoG is recognized by Gly42 and the Watson-Crick edge of oxoG is contacted by Gln315 and Pro266. The previously observed salt bridge between Lys249 and Cys253 is lacking in the FLRC, suggesting Lys249 is primed by Cys253 and poised for nucleophilic attack on C1' of oxodG. Overall, hOGG1 FLRC marks the first structure of oxoG presented into an intact catalytic site of hOGG1 and provides complementary insights into the glycosylase mechanisms of the enzyme.
Assuntos
DNA Glicosilases , Humanos , Aminoácidos/metabolismo , Domínio Catalítico , DNA/química , Dano ao DNA , DNA Glicosilases/metabolismo , Reparo do DNA , DNA-Formamidopirimidina Glicosilase/genética , DNA-Formamidopirimidina Glicosilase/metabolismo , Guanina/metabolismo , Estresse OxidativoRESUMO
RNA abasic sites and the mechanisms involved in their regulation are mostly unknown; in contrast, DNA abasic sites are well-studied. We found surprisingly that, in yeast and human cells, RNA abasic sites are prevalent. When a base is lost from RNA, the remaining ribose is found as a closed-ring or an open-ring sugar with a reactive C1' aldehyde group. Using primary amine-based reagents that react with the aldehyde group, we uncovered evidence for abasic sites in nascent RNA, messenger RNA, and ribosomal RNA from yeast and human cells. Mass spectroscopic analysis confirmed the presence of RNA abasic sites. The RNA abasic sites were found to be coupled to R-loops. We show that human methylpurine DNA glycosylase cleaves N-glycosidic bonds on RNA and that human apurinic/apyrimidinic endonuclease 1 incises RNA abasic sites in RNA-DNA hybrids. Our results reveal that, in yeast and human cells, there are RNA abasic sites, and we identify a glycosylase that generates these sites and an AP endonuclease that processes them.
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Sequência de Bases/genética , RNA/química , RNA/genética , Sítios de Ligação , DNA/química , Dano ao DNA/genética , DNA Glicosilases/metabolismo , Reparo do DNA/genética , DNA Liase (Sítios Apurínicos ou Apirimidínicos)/genética , Desoxirribonuclease I/metabolismo , Humanos , Nucleotídeos/genética , Estruturas R-Loop/genética , Saccharomyces cerevisiae/genética , Especificidade por Substrato , Leveduras/genéticaRESUMO
This research is about the profiling of barley (Hordeum vulgare L.), wheat (Triticum aestivum L.), and rye (Secale cereale L.) FPG and OGG1 genes during grain germination. During seed germination, reactive oxygen species accumulate, which leads to DNA damage. In the base excision repair (BER) system, the enzymes formamidopyrimidine DNA glycosylase (FPG) and 8-oxoguanine DNA glycosylase (OGG1), among others, are responsible for repairing such damage. We decided to check how the expression of genes encoding these two enzymes changes in germinating grains. Spring varieties of barley, wheat, and rye from the previous growing season were used in the study. Expression level changes were checked using Real-Time PCR. After analyzing the obtained results, the maximum expression levels of FPG and OGG1 genes during germination were determined for barley, wheat, and rye. The results of the study show differences in expression levels specific to each species. The highest expression was observed at different time points for each of them. There were no differences in the highest expression for FPG and OGG1 within one species. In conclusion, the research provides information on how the level of FPG and OGG1 gene expression changes during the germination process in cereals. This is the first study looking at the expression levels of these two genes in cereals.
Assuntos
Hordeum , DNA-Formamidopirimidina Glicosilase , Hordeum/genética , Triticum/genética , Grão Comestível/genética , Secale/genética , Germinação/genéticaRESUMO
The protein encoded by the vaccinia virus D4R gene has base excision repair uracil-DNA N-glycosylase (vvUNG) activity and also acts as a processivity factor in the viral replication complex. The use of a protein unlike PolN/PCNA sliding clamps is a unique feature of orthopoxviral replication, providing an attractive target for drug design. However, the intrinsic processivity of vvUNG has never been estimated, leaving open the question whether it is sufficient to impart processivity to the viral polymerase. Here, we use the correlated cleavage assay to characterize the translocation of vvUNG along DNA between two uracil residues. The salt dependence of the correlated cleavage, together with the similar affinity of vvUNG for damaged and undamaged DNA, support the one-dimensional diffusion mechanism of lesion search. Unlike short gaps, covalent adducts partly block vvUNG translocation. Kinetic experiments show that once a lesion is found it is excised with a probability ~0.76. Varying the distance between two uracils, we use a random walk model to estimate the mean number of steps per association with DNA at ~4200, which is consistent with vvUNG playing a role as a processivity factor. Finally, we show that inhibitors carrying a tetrahydro-2,4,6-trioxopyrimidinylidene moiety can suppress the processivity of vvUNG.
Assuntos
Uracila-DNA Glicosidase , Vaccinia virus , Uracila-DNA Glicosidase/genética , Uracila-DNA Glicosidase/metabolismo , Vaccinia virus/genética , DNA/metabolismo , Replicação Viral , Reparo do DNA , Uracila/metabolismo , DNA Viral/genética , DNA Viral/metabolismo , Replicação do DNARESUMO
Adenine-DNA-glycosylase MutY is a monofunctional enzyme and catalyzes hydrolysis of N-glycosidic bonds with adenine residues located opposite 8-oxonuanine residues in DNA. Rational design was carried out to construct mutant enzyme forms with altered catalytic activity. Structures of the MutY mutants were calculated by molecular dynamics (MD). Their analysis showed that some of the MutY mutants may have AP lyase activity in addition to hydrolyzing the N-glycosidic bond, as is the case with bifunctional DNA glycosylases. MutY mutants with the A120K or S124K substitution were obtained by site-directed mutagenesis, and their catalytic activities were determined. The S120K substitution was shown to confer additional AP lyase activity, while the A124K substitution completely inactivated the enzyme.
Assuntos
Reparo do DNA , DNA Liase (Sítios Apurínicos ou Apirimidínicos) , DNA Liase (Sítios Apurínicos ou Apirimidínicos)/genética , Escherichia coli/genética , Escherichia coli/metabolismo , Engenharia de Proteínas , DNA/química , Adenina/química , Adenina/metabolismoRESUMO
The important role of DNA damage in the occurrence of various diseases, including cancer, has led to study of the mechanisms of genetic information stability, that have been carried out since the discovery of DNA repair systems. The question of the relationship between the accumulation of DNA damage, disorders in DNA repair pathways, and increased risk of disease development is still relevant. Over the past few years, significant efforts have been made to develop methods for analyzing the activity of DNA repair enzymes in human cells. In this work, we developed fluorescent DNA probes that allow us to determine the activity of key enzymes of base excision DNA repair in cell extracts, namely the DNA glycosylases UNG2, SMUG1, MBD4, TDG, AAG, NEIL1, NTHL1, and OGG1 and the AP endonuclease APE1. The sensitivity of DNA probes was determined on pure enzyme preparations. Determination of the activity of repair enzymes in cell extracts of the human ovarian tumor lines TOV112, 79, OVCAR3, MESOV, SCOV3, and TOV21 revealed significant variability in the level of enzyme activity in these cell lines. These results may become a test system platform for analyzing the activity of the base excision DNA repair system in the human body.