Your browser doesn't support javascript.
loading
Mostrar: 20 | 50 | 100
Resultados 1 - 20 de 377
Filtrar
1.
Talanta ; 235: 122805, 2021 Dec 01.
Artigo em Inglês | MEDLINE | ID: mdl-34517663

RESUMO

DNA glycosylases can initiate base excision repair pathway to repair endogenous DNA base damages for the maintenance of genome stability. Multiple DNA glycosylases exhibit abnormal in various diseases, and the simultaneous measurement of different DNA glycosylases is critical to clinical diagnosis and drug discovery. Herein, we take advantage of single-molecule detection and bidirectional strand displacement amplification (SDA) to simultaneously detect uracil DNA glycolase (UDG) and human alkyladenine DNA glycosylase (hAAG). We design a partial double-stranded DNA (dsDNA) substrate modified with specific recognition sites of UDG and hAAG. The dsDNA substrate is labeled with BHQ1 and BHQ2 at the 5'-ends and then hybridizes with the Cy3/Cy5-labeled reporter probes to obtain the BHQ1/Cy3 and BHQ2/Cy5 base pairs, resulting in the quenching of Cy3/Cy5 fluorescence by BHQ1/BHQ2 via fluorescence resonance energy transfer (FRET). When UDG and hAAG are present, they can induce the base excision repair reaction and subsequently initiate the bidirectional SDA amplification process, releasing the Cy5/Cy3-labeled reporter probes from the dsDNA substrate and consequently the recovery of Cy5 and Cy3 fluorescence, which can be measured by single-molecule detection, with Cy5 indicating UDG and Cy3 indicating hAAG. This method possesses high sensitivity and good selectivity with the capability of quantifying multiple DNA glycosylases at the single-cell level. Furthermore, it can be used to simultaneously screen DNA glycosylase inhibitors and determine enzyme kinetic parameters, with the potential of sensing various DNA/RNA enzymes by simple changing the recognition sites of DNA substrates.


Assuntos
Reparo do DNA , Uracila-DNA Glicosidase , Bioensaio , DNA/genética , Transferência Ressonante de Energia de Fluorescência , Humanos , Uracila-DNA Glicosidase/metabolismo
2.
Molecules ; 26(17)2021 Aug 26.
Artigo em Inglês | MEDLINE | ID: mdl-34500606

RESUMO

Ionizing radiation is a factor that seriously damages cellular mechanisms/macromolecules, e.g., by inducing damage in the human genome, such as 5',8-cyclo-2'-deoxypurines (cdPus). CdPus may become a component of clustered DNA lesions (CDL), which are notably unfavorable for the base excision repair system (BER). In this study, the influence of 5'S and 5'R diastereomers of 5',8-cyclo-2'-deoxyadenosine (cdA) and 5',8-cyclo-2'-deoxyguanosine (cdG) on the uracil-DNA glycosylase (UDG) and human AP site endonuclease 1 (hAPE1) activity has been taken under consideration. Synthetic oligonucleotides containing 2'-deoxyuridine (dU) and cdPu were used as a model of single-stranded CDL. The activity of the UDG and hAPE1 enzymes decreased in the presence of RcdG compared to ScdG. Contrary to the above, ScdA reduced enzyme activity more than RcdA. The presented results show the influence of cdPus lesions located within CDL on the activity of the initial stages of BER dependently on their position toward dU. Numerous studies have shown the biological importance of cdPus (e.g., as a risk of carcinogenesis). Due to that, it is important to understand how to recognize and eliminate this type of DNA damage from the genome.


Assuntos
DNA Liase (Sítios Apurínicos ou Apirimidínicos)/metabolismo , Desoxiadenosinas/metabolismo , Desoxiguanosina/metabolismo , Uracila-DNA Glicosidase/metabolismo , DNA/genética , DNA/metabolismo , Dano ao DNA/genética , Reparo do DNA/genética , Humanos , Oligonucleotídeos/metabolismo
3.
Nat Commun ; 12(1): 4902, 2021 08 12.
Artigo em Inglês | MEDLINE | ID: mdl-34385461

RESUMO

Efficient and precise base editors (BEs) for C-to-G transversion are highly desirable. However, the sequence context affecting editing outcome largely remains unclear. Here we report engineered C-to-G BEs of high efficiency and fidelity, with the sequence context predictable via machine-learning methods. By changing the species origin and relative position of uracil-DNA glycosylase and deaminase, together with codon optimization, we obtain optimized C-to-G BEs (OPTI-CGBEs) for efficient C-to-G transversion. The motif preference of OPTI-CGBEs for editing 100 endogenous sites is determined in HEK293T cells. Using a sgRNA library comprising 41,388 sequences, we develop a deep-learning model that accurately predicts the OPTI-CGBE editing outcome for targeted sites with specific sequence context. These OPTI-CGBEs are further shown to be capable of efficient base editing in mouse embryos for generating Tyr-edited offspring. Thus, these engineered CGBEs are useful for efficient and precise base editing, with outcome predictable based on sequence context of targeted sites.


Assuntos
Sistemas CRISPR-Cas , Citidina Desaminase/metabolismo , Edição de Genes/métodos , Aprendizado de Máquina , Uracila-DNA Glicosidase/metabolismo , Animais , Sequência de Bases , Sítios de Ligação/genética , Caenorhabditis elegans/genética , Códon/genética , Citidina Desaminase/genética , Escherichia coli/genética , Feminino , Biblioteca Gênica , Células HEK293 , Humanos , Camundongos , Reprodutibilidade dos Testes , Uracila-DNA Glicosidase/genética
4.
Talanta ; 234: 122680, 2021 Nov 01.
Artigo em Inglês | MEDLINE | ID: mdl-34364480

RESUMO

Uracil DNA glycosylase (UDG) is a key base excision repair (BER) enzyme and its abnormal expression is nearly relevant to several diseases including cancer. The sensitive detection of UDG activity is beneficial for biomedical studies and clinic diagnosis. In this work, we proposed a dumbbell probe mediated triple cascade signal amplification strategy for sensitive and specific detection of UDG activity. The specially designed dumbbell probe contained two uracil bases, two recognition sites for nicking enzyme and a split sequence of DNAzyme. Unsealed dumbbell probes were first connected into sealed dumbbell probes by T4 DNA ligase, and then the unsealed probes were hydrolyzed by exonuclease to ensure the purity of probes. Under the influence of UDG, two uracil bases were removed to produce two apyrimidinic (AP) sites, which were subsequently cleaved by Endo.IV. The probes after cleavage acted as primers and templates for double nicking sites strand displacement amplification (SDA) to produce a mass of two products. The products of SDA continued to act as primers and templates for rolling circle amplification (RCA) to produce repeats containing complete DNAzyme sequences. The DNAzyme repeatedly cleaved multiple molecular beacons (MB), resulting in remarkable fluorescence enhancement. Benefiting from the triple cascade signal amplification, the sensitivity was improved and the detection limit was 7.2 × 10-5 U mL-1. The method could well distinguish UDG from other interfering enzymes and detect UDG activity in real biological samples, showing good specificity. In addition, this method could be used for screening inhibitors. The above results suggested that the method provided a promising analytical means for UDG related biomedical research and clinic diagnosis.


Assuntos
DNA Catalítico , Uracila-DNA Glicosidase , Reparo do DNA , Fluorescência , Humanos , Uracila , Uracila-DNA Glicosidase/metabolismo
5.
Appl Microbiol Biotechnol ; 105(13): 5449-5460, 2021 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-34223949

RESUMO

Genomes of hyperthermophiles are facing a severe challenge due to increased deamination rates of cytosine induced by high temperature, which could be counteracted by base excision repair mediated by uracil DNA glycosylase (UDG) or other repair pathways. Our previous work has shown that the two UDGs (Tba UDG247 and Tba UDG194) encoded by the genome of the hyperthermophilic euryarchaeon Thermococcus barophilus Ch5 can remove uracil from DNA at high temperature. Herein, we provide evidence that Tba UDG247 is a novel bifunctional glycosylase which can excise uracil from DNA and further cleave the phosphodiester bo nd of the generated apurinic/apyrimidinic (AP) site, which has never been described to date. In addition to cleaving uracil-containing DNA, Tba UDG247 can also cleave AP-containing ssDNA although at lower efficiency, thereby suggesting that the enzyme might be involved in repair of AP site in DNA. Kinetic analyses showed that Tba UDG247 displays a faster rate for uracil excision than for AP cleavage, thus suggesting that cleaving AP site by the enzyme is a rate-limiting step for its bifunctionality. Phylogenetic analysis showed that Tba UDG247 is clustered on a separate branch distant from all the reported UDGs. Overall, we designated Tba UDG247 as the prototype of a novel family of bifunctional UDGs. KEY POINTS: We first reported a novel DNA glycosylase with bifunctionality. Tba UDG247 possesses an AP lyase activity.


Assuntos
Thermococcus , Reparo do DNA , Filogenia , Thermococcus/genética , Thermococcus/metabolismo , Uracila , Uracila-DNA Glicosidase/genética , Uracila-DNA Glicosidase/metabolismo
6.
Viruses ; 13(5)2021 05 10.
Artigo em Inglês | MEDLINE | ID: mdl-34068736

RESUMO

Deoxyuridine in DNA has recently been in the focus of research due to its intriguing roles in several physiological and pathophysiological situations. Although not an orthodox DNA base, uracil may appear in DNA via either cytosine deamination or thymine-replacing incorporations. Since these alterations may induce mutation or may perturb DNA-protein interactions, free living organisms from bacteria to human contain several pathways to counteract uracilation. These efficient and highly specific repair routes uracil-directed excision repair initiated by representative of uracil-DNA glycosylase families. Interestingly, some bacteriophages exist with thymine-lacking uracil-DNA genome. A detailed understanding of the strategy by which such phages can replicate in bacteria where an efficient repair pathway functions for uracil-excision from DNA is expected to reveal novel inhibitors that can also be used for biotechnological applications. Here, we also review the several potential biotechnological applications already implemented based on inhibitors of uracil-excision repair, such as Crispr-base-editing and detection of nascent uracil distribution pattern in complex genomes.


Assuntos
DNA Viral/química , DNA Viral/genética , Uracila , Vírus/genética , Bacteriófagos/efeitos dos fármacos , Bacteriófagos/genética , Bacteriófagos/metabolismo , Biotecnologia , DNA Viral/metabolismo , Desenvolvimento de Medicamentos , Humanos , Modelos Moleculares , Ácidos Nucleicos/química , Ácidos Nucleicos/metabolismo , Conformação Proteica , Relação Estrutura-Atividade , Uracila/química , Uracila-DNA Glicosidase/química , Uracila-DNA Glicosidase/metabolismo , Vírus/efeitos dos fármacos , Vírus/metabolismo
7.
Talanta ; 232: 122429, 2021 Sep 01.
Artigo em Inglês | MEDLINE | ID: mdl-34074415

RESUMO

DNA glycosylase is an indispensable DNA damage repair enzyme which can recognize and excise the damaged bases in the DNA base excision-repair pathway. The dysregulation of DNA glycosylase activity will give rise to the dysfunction of base excision-repair and lead to abnormalities and diseases. The simultaneous detection of multiple DNA glycosylases can help to fully understand the normal physiological functions of cells, and determine whether the cells are abnormal in pre-disease. Regrettably, the synchronous detection of functionally similar DNA glycosylases is a great challenge. Herein, we developed a multifunctional dsDNA probe mediated exponential rolling circle amplification (E-RCA) method for the simultaneously sensitive detection of human alkyladenine DNA glycosylase (hAAG) and uracil-DNA glycosylase (UDG). The multifunctional dsDNA probe contains the hypoxanthine sites and the uracil sites which can be recognized by hAAG and UDG respectively to generate apyrimidinic (AP) sites in the dsDNA probe. Then the AP sites will be recognized and cut by endonuclease Ⅳ (Endo IV) to release corresponding single-stranded primer probes. Subsequently, two padlock DNA templates are added to initiate E-RCA to generate multitudinous G-quadruplexes and/or double-stranded dumbbell lock structures, which can combine N-methyl mesoporphyrin IX (NMM) and SYBR Green Ⅰ (SGI) for the generation of respective fluorescent signals. The detection limits are obtained as low as 0.0002 U mL-1 and 0.00001 U mL-1 for hAAG and UDG, respectively. Notably, this method can realize the simultaneous detection of two DNA glycosylases without the use of specially labeled probes. Finally, this method is successfully applied to detect hAAG and UDG activities in the lysates of HeLa cells and Endo1617 cells at single-cell level, and to detect the inhibitors of DNA glycosylases.


Assuntos
DNA Glicosilases , Técnicas de Amplificação de Ácido Nucleico , Uracila-DNA Glicosidase , Sondas de DNA , Reparo do DNA , Células HeLa , Humanos , Limite de Detecção , Uracila-DNA Glicosidase/metabolismo
8.
Anal Chem ; 93(18): 6913-6918, 2021 05 11.
Artigo em Inglês | MEDLINE | ID: mdl-33929831

RESUMO

Human 8-oxoguanine DNA glycosylase (hOGG1) can initiate base excision repair of genomic 8-oxoguanine (8-oxoG), and it can locate and remove damaged 8-oxoG through extrusion and excision. Sensitive detection of hOGG1 is critical for clinical diagnosis. Herein, we develop a simple mix-and-read assay for the sensitive detection of DNA glycosylase using multiple cyclic enzymatic repairing amplification. The hOGG1 can excise the 8-oxoG base of the DNA substrate to produce an apurinic/apyrimidinic (AP) site, and then, the AP site can be cleaved by apurinic/apyrimidic endonuclease 1 (APE1), producing the substrate fragment with a free 3'-OH terminus. Subsequently, the substrate fragment can initiate cyclic enzymatic repairing amplification, generating two triggers. The resultant two triggers can function as the primers to induce three cyclic enzymatic repairing amplification, respectively, producing more and more triggers. We experimentally verify the occurrence of each cyclic enzymatic repairing amplification and uracil DNA glycosylase (UDG)-mediated exponential amplification. The amplification products can be simply detected using SYBR Green II as the fluorescent dye. This mix-and-read assay is very simple and rapid (within 40 min) without the requirement of any extra primers and modification/separation steps. This method can sensitively measure hOGG1 with a detection limit of 2.97 × 10-8 U/µL, and it can be applied for the screening of inhibitors and the monitoring of cellular hOGG1 activity at the single-cell level, providing an adaptive and flexible tool for clinical diagnosis and drug discovery.


Assuntos
DNA Liase (Sítios Apurínicos ou Apirimidínicos) , Uracila-DNA Glicosidase , DNA , Reparo do DNA , DNA Liase (Sítios Apurínicos ou Apirimidínicos)/metabolismo , Corantes Fluorescentes , Humanos , Uracila-DNA Glicosidase/metabolismo
9.
Anal Biochem ; 623: 114193, 2021 06 15.
Artigo em Inglês | MEDLINE | ID: mdl-33831350

RESUMO

Technological advancements have revolutionized ancient and degraded DNA analysis, moving the field to the Next Generation Sequencing era. One of the advancements, the ancient DNA-oriented high-throughput library preparation methods, enabled the sequencing of more endogenous molecules. Although fairly optimized, both single- and double-stranded library preparation methods hold the potential for further improvement. Here, we test a series of modifications made at different steps of both single- and double-stranded library preparation methods. Given all the modifications tested, we found that two of them provide further benefits, including the use of Endonuclease VIII as a pre-treatment step before preparing single-stranded libraries and the use of a modified second adapter of the single stranded-libraries as an alternative option to enable sequencing of single stranded-libraries with the standard Illumina sequencing primer instead of the custom designed as described in the single stranded library preparation method. Furthermore, we propose uracil-DNA-glycosylase (UDG) could also be considered for both single- and double-stranded library preparation methods, although additional parameters should be taken into account depending on the sequencing strategy and the sample characteristics. Further modifications were also tested and although they were not advantageous, they could be considered as equivalent to the published options.


Assuntos
DNA Antigo/análise , DNA/análise , Biblioteca Genômica , Sequenciamento de Nucleotídeos em Larga Escala/métodos , Análise de Sequência de DNA/métodos , DNA/química , DNA/metabolismo , Primers do DNA/química , DNA Antigo/química , DNA de Cadeia Simples/análise , DNA de Cadeia Simples/química , Humanos , Uracila-DNA Glicosidase/química , Uracila-DNA Glicosidase/metabolismo
10.
Mol Pharmacol ; 99(6): 412-425, 2021 06.
Artigo em Inglês | MEDLINE | ID: mdl-33795350

RESUMO

Previous short-hairpin RNA knockdown studies have established that depletion of human uracil DNA glycosylase (hUNG) sensitizes some cell lines to 5-fluorodeoxyuridine (FdU). Here, we selectively inhibit the catalytic activity of hUNG by lentiviral transduction of uracil DNA glycosylase inhibitor protein into a large panel of cancer cell lines under control of a doxycycline-inducible promoter. This induced inhibition strategy better assesses the therapeutic potential of small-molecule targeting of hUNG. In total, 6 of 11 colorectal lines showed 6- to 70-fold increases in FdU potency upon hUNG inhibition ("responsive"). This hUNG-dependent response was not observed with fluorouracil (FU), indicating that FU does not operate through the same DNA repair mechanism as FdU in vitro. Potency of the thymidylate synthase inhibitor raltitrexed (RTX), which elevates deoxyuridine triphosphate levels, was only incrementally enhanced upon hUNG inhibition (<40%), suggesting that responsiveness is associated with incorporation and persistence of FdU in DNA rather than deoxyuridine. The importance of FU/A and FU/G lesions in the toxicity of FdU is supported by the observation that dT supplementation completely rescued the toxic effects of U/A lesions resulting from RTX, but dT only increased the IC50 for FdU, which forms both FU/A and FU/G mismatches. Contrary to previous reports, cellular responsiveness to hUNG inhibition did not correlate with p53 status or thymine DNA glycosylase expression. A model is suggested in which the persistence of FU/A and FU/G base pairs in the absence of hUNG activity elicits an apoptotic DNA damage response in both responsive and nonresponsive colorectal lines. SIGNIFICANCE STATEMENT: The pyrimidine base 5-fluorouracil is a mainstay chemotherapeutic for treatment of advanced colorectal cancer. Here, this study shows that its deoxynucleoside form, 5-fluorodeoxyuridine (FdU), operates by a distinct DNA incorporation mechanism that is strongly potentiated by inhibition of the DNA repair enzyme human uracil DNA glycosylase. The hUNG-dependent mechanism was present in over 50% of colorectal cell lines tested, suggesting that a significant fraction of human cancers may be sensitized to FdU in the presence of a small-molecule hUNG inhibitor.


Assuntos
Antineoplásicos/farmacologia , Neoplasias Colorretais/patologia , Floxuridina/farmacologia , Fluoruracila/farmacologia , Quinazolinas/farmacologia , Tiofenos/farmacologia , Uracila-DNA Glicosidase/antagonistas & inibidores , Linhagem Celular Tumoral , Dano ao DNA , Ensaios de Seleção de Medicamentos Antitumorais , Humanos , Uracila-DNA Glicosidase/metabolismo
11.
Science ; 372(6538): 156-165, 2021 04 09.
Artigo em Inglês | MEDLINE | ID: mdl-33833118

RESUMO

Mutations in the BRCA1 or BRCA2 tumor suppressor genes predispose individuals to breast and ovarian cancer. In the clinic, these cancers are treated with inhibitors that target poly(ADP-ribose) polymerase (PARP). We show that inhibition of DNPH1, a protein that eliminates cytotoxic nucleotide 5-hydroxymethyl-deoxyuridine (hmdU) monophosphate, potentiates the sensitivity of BRCA-deficient cells to PARP inhibitors (PARPi). Synthetic lethality was mediated by the action of SMUG1 glycosylase on genomic hmdU, leading to PARP trapping, replication fork collapse, DNA break formation, and apoptosis. BRCA1-deficient cells that acquired resistance to PARPi were resensitized by treatment with hmdU and DNPH1 inhibition. Because genomic hmdU is a key determinant of PARPi sensitivity, targeting DNPH1 provides a promising strategy for the hypersensitization of BRCA-deficient cancers to PARPi therapy.


Assuntos
Antineoplásicos/farmacologia , N-Glicosil Hidrolases/antagonistas & inibidores , N-Glicosil Hidrolases/metabolismo , Inibidores de Poli(ADP-Ribose) Polimerases/farmacologia , Proteínas Proto-Oncogênicas/antagonistas & inibidores , Proteínas Proto-Oncogênicas/metabolismo , Apoptose , Sistemas CRISPR-Cas , Linhagem Celular Tumoral , Quebras de DNA de Cadeia Dupla , Replicação do DNA , DNA de Neoplasias/metabolismo , Desoxicitidina Monofosfato/análogos & derivados , Desoxicitidina Monofosfato/metabolismo , Desoxicitidina Monofosfato/farmacologia , Nucleotídeos de Desoxiuracil/metabolismo , Resistencia a Medicamentos Antineoplásicos , Genes BRCA1 , Humanos , Hidrólise , N-Glicosil Hidrolases/genética , Ftalazinas/farmacologia , Piperazinas/farmacologia , Poli(ADP-Ribose) Polimerases/metabolismo , Proteínas Proto-Oncogênicas/genética , Mutações Sintéticas Letais , Timidina/análogos & derivados , Timidina/antagonistas & inibidores , Timidina/metabolismo , Timidina/farmacologia , Uracila-DNA Glicosidase/metabolismo
12.
Int J Mol Sci ; 22(8)2021 Apr 09.
Artigo em Inglês | MEDLINE | ID: mdl-33918885

RESUMO

The appearance of uracil in the deoxyuridine moiety of DNA is among the most frequently occurring genomic modifications. Three different routes can result in genomic uracil, two of which do not require specific enzymes: spontaneous cytosine deamination due to the inherent chemical reactivity of living cells, and thymine-replacing incorporation upon nucleotide pool imbalances. There is also an enzymatic pathway of cytosine deamination with multiple DNA (cytosine) deaminases involved in this process. In order to describe potential roles of genomic uracil, it is of key importance to utilize efficient uracil-DNA detection methods. In this review, we provide a comprehensive and critical assessment of currently available uracil detection methods with special focus on genome-wide mapping solutions. Recent developments in PCR-based and in situ detection as well as the quantitation of genomic uracil are also discussed.


Assuntos
DNA , Genoma , Uracila , Animais , DNA/química , DNA/metabolismo , Reparo do DNA , Replicação do DNA , Testes Genéticos , Estudo de Associação Genômica Ampla , Sequenciamento de Nucleotídeos em Larga Escala , Humanos , Hibridização In Situ , Nucleotídeos , Reação em Cadeia da Polimerase , Transdução de Sinais , Uracila/química , Uracila/metabolismo , Uracila-DNA Glicosidase/metabolismo
13.
Mol Biol (Mosk) ; 55(2): 277-288, 2021.
Artigo em Russo | MEDLINE | ID: mdl-33871441

RESUMO

The human N-glycosylases SMUG1 and MBD4 catalyze the removal of uracil residues from DNA resulting from cytosine deamination or replication errors. For polymorphic variants of SMUG1 (G90C, P240H, N244S, N248Y) and the MBD4^(cat) catalytic domain (S470L, G507S, R512W, H557D), the structures of enzyme-substrate complexes were obtained by molecular dynamic simulation. It was experimentally found that the SNP variants of SMUG1, N244S and N248Y, had increased catalytic activity compared to the wild-type enzyme, probably due to the acceleration of the dissociation of the enzyme-product complex and an increase in the enzyme turnover rate. All other SNP variants of SMUG1 (G90C, P240H) and MBD4^(cat), in which amino acid substitutions disrupted the substrate binding region and/or active site, had significantly lower catalytic activity than the wild-type enzymes.


Assuntos
Reparo do DNA , Uracila-DNA Glicosidase , DNA , Dano ao DNA , Endodesoxirribonucleases , Humanos , Uracila , Uracila-DNA Glicosidase/genética , Uracila-DNA Glicosidase/metabolismo
14.
Int J Mol Sci ; 22(4)2021 Feb 17.
Artigo em Inglês | MEDLINE | ID: mdl-33671338

RESUMO

Single-strand selective monofunctional uracil DNA glycosylase 1 (SMUG1) works to remove uracil and certain oxidized bases from DNA during base excision repair (BER). This review provides a historical characterization of SMUG1 and 5-hydroxymethyl-2'-deoxyuridine (5-hmdU) one important substrate of this enzyme. Biochemical and structural analyses provide remarkable insight into the mechanism of this glycosylase: SMUG1 has a unique helical wedge that influences damage recognition during repair. Rodent studies suggest that, while SMUG1 shares substrate specificity with another uracil glycosylase UNG2, loss of SMUG1 can have unique cellular phenotypes. This review highlights the multiple roles SMUG1 may play in preserving genome stability, and how the loss of SMUG1 activity may promote cancer. Finally, we discuss recent studies indicating SMUG1 has moonlighting functions beyond BER, playing a critical role in RNA processing including the RNA component of telomerase.


Assuntos
Genoma , Neoplasias/enzimologia , Neoplasias/genética , Uracila-DNA Glicosidase/metabolismo , Animais , Citoproteção , Humanos , Nucleossomos/metabolismo , Especificidade por Substrato
15.
Chem Commun (Camb) ; 57(22): 2784-2787, 2021 Mar 16.
Artigo em Inglês | MEDLINE | ID: mdl-33599665

RESUMO

We report a new method for dU detection in genomic DNA combined with UNG excision and fluorescent probe labeling. UNG can remove uracil bases to introduce abasic sites, which can react with NRNO to produce intense fluorescence because of the inhibition of the PET effect. It can also cause the polymerase extension to stop to provide details of dU site information.


Assuntos
DNA/química , Corantes Fluorescentes/química , Genômica , Uracila/análise , Reparo do DNA , Mutação , Uracila-DNA Glicosidase/metabolismo
16.
DNA Repair (Amst) ; 101: 103077, 2021 05.
Artigo em Inglês | MEDLINE | ID: mdl-33640758

RESUMO

The presence of uracil in DNA calls for rapid removal facilitated by the uracil-DNA glycosylase superfamily of enzymes, which initiates the base excision repair (BER) pathway. In humans, uracil excision is accomplished primarily by the human uracil-DNA glycosylase (hUNG) enzymes. In addition to BER, hUNG enzymes play a key role in somatic hypermutation to generate antibody diversity. hUNG has several isoforms, with hUNG1 and hUNG2 being the two major isoforms. Both isoforms contain disordered N-terminal domains, which are responsible for a wide range of functions, with minimal direct impact on catalytic efficiency. Subcellular localization of hUNG enzymes is directed by differing N-terminal sequences, with hUNG1 dedicated to mitochondria and hUNG2 dedicated to the nucleus. An alternative isoform of hUNG1 has also been identified to localize to the nucleus in mouse and human cell models. Furthermore, hUNG2 has been observed at replication forks performing both pre- and post-replicative uracil excision to maintain genomic integrity. Replication protein A (RPA) and proliferating cell nuclear antigen (PCNA) are responsible for recruitment to replication forks via protein-protein interactions with the N-terminus of hUNG2. These interactions, along with protein degradation, are regulated by various post-translational modifications within the N-terminal tail, which are primarily cell-cycle dependent. Finally, translocation on DNA is also mediated by interactions between the N-terminus and DNA, which is enhanced under molecular crowding conditions by preventing diffusion events and compacting tail residues. This review summarizes recent research supporting the emerging roles of the N-terminal domain of hUNG.


Assuntos
Reparo do DNA , Domínios Proteicos , Uracila-DNA Glicosidase/metabolismo , Animais , DNA/metabolismo , Dano ao DNA , Humanos , Ligação Proteica , Isoformas de Proteínas , Processamento de Proteína Pós-Traducional
17.
Biomolecules ; 11(1)2021 01 08.
Artigo em Inglês | MEDLINE | ID: mdl-33430019

RESUMO

RNA modifications are essential for proper RNA processing, quality control, and maturation steps. In the last decade, some eukaryotic DNA repair enzymes have been shown to have an ability to recognize and process modified RNA substrates and thereby contribute to RNA surveillance. Single-strand-selective monofunctional uracil-DNA glycosylase 1 (SMUG1) is a base excision repair enzyme that not only recognizes and removes uracil and oxidized pyrimidines from DNA but is also able to process modified RNA substrates. SMUG1 interacts with the pseudouridine synthase dyskerin (DKC1), an enzyme essential for the correct assembly of small nucleolar ribonucleoproteins (snRNPs) and ribosomal RNA (rRNA) processing. Here, we review rRNA modifications and RNA quality control mechanisms in general and discuss the specific function of SMUG1 in rRNA metabolism. Cells lacking SMUG1 have elevated levels of immature rRNA molecules and accumulation of 5-hydroxymethyluridine (5hmU) in mature rRNA. SMUG1 may be required for post-transcriptional regulation and quality control of rRNAs, partly by regulating rRNA and stability.


Assuntos
Processamento Pós-Transcricional do RNA , RNA Ribossômico/metabolismo , Uracila-DNA Glicosidase/metabolismo , Animais , Humanos , Modelos Moleculares , Estabilidade de RNA , RNA Ribossômico/química
18.
Biosens Bioelectron ; 171: 112734, 2021 Jan 01.
Artigo em Inglês | MEDLINE | ID: mdl-33069955

RESUMO

A simple and highly sensitive biosensing strategy was reported by cascading terminal deoxynucleotidyl transferase (TdT)-catalyzed substrate extension and CRISPR-Cas12a -catalyzed short-stranded DNA probe cleavage. Such a strategy, which is named as TdT-combined CRISPR-Cas12a amplification, gives excellent signal amplification capability due to the synergy of two amplification steps, and thus shows great promise in the design of various biosensors. Based on this strategy, two representative biosensors were developed by simply adjusting the DNA substrate design. High signal amplification efficiency and nearly zero background endowed the biosensors with extraordinary high sensitivity. By utilizing these two biosensors, ultrasensitive detection of uracil-DNA glycosylase (UDG) and T4 polynucleotide kinase (T4 PNK) was achieved with the detection limit as low as 5 × 10-6 U/mL and 1 × 10-4 U/mL, respectively. The proposed UDG-sensing platform was also demonstrated to work well for the UDG activity detection in cancer cells as well as UDG screening and inhibitory capability evaluation, thus showing a great potential in clinical diagnosis and biomedical research.


Assuntos
Técnicas Biossensoriais , Uracila-DNA Glicosidase , Sistemas CRISPR-Cas , Repetições Palindrômicas Curtas Agrupadas e Regularmente Espaçadas , DNA Nucleotidilexotransferase , Uracila-DNA Glicosidase/genética , Uracila-DNA Glicosidase/metabolismo
19.
Talanta ; 221: 121609, 2021 Jan 01.
Artigo em Inglês | MEDLINE | ID: mdl-33076139

RESUMO

Uracil-DNA glycosylase (UDG) is a crucial enzyme in base excision repair (BER) pathway. It can repair the uracil-induced DNA lesions and maintain the integrity of genome. In this paper, we developed a facile and ratiometric strategy for UDG activity detection using fluorescence resonance energy transfer (FRET). One double-stranded DNA (dsDNA) substrate consisting of strand 1 (dual-fluorescent dye-modified G-quadruplex sequence single-stranded DNA (ssDNA)), carboxyfluorescein (FAM) acted as donor and tetramethylrhodamine (TAMRA) as acceptor) and strand 2 (the complementary sequence of strand 1 containing three mismatched bases and three uracil bases) was introduced. When the UDG-catalyzed uracil is removed from dsDNA, the thermo-stability of dsDNA is decreased and the dual-fluorescent dye-modified G-quadruplex sequence ssDNA is released. Then, the ssDNA transforms into a G-quadruplex comformation, which brings the labeled FAM and TAMRA into close proximity, resulting in a strong FRET signal. In the absence of UDG, the relatively stable dsDNA separates the labeled FAM and TAMRA, giving a weak FRET signal. Thus, by measuring the system fluorescence intensity and exploiting FRET signal difference, UDG activity can be detected in a simple process. The detection limit is 0.087 U/mL without requiring additional signal amplification process. Besides, our developed strategy can also be used for screening the UDG inhibitors in a ratiometric fluorescence detection way.


Assuntos
Quadruplex G , Uracila-DNA Glicosidase , DNA , Reparo do DNA , Transferência Ressonante de Energia de Fluorescência , Uracila-DNA Glicosidase/metabolismo
20.
PLoS Genet ; 16(12): e1008960, 2020 12.
Artigo em Inglês | MEDLINE | ID: mdl-33362210

RESUMO

Most B cell lymphomas originate from B cells that have germinal center (GC) experience and bear chromosome translocations and numerous point mutations. GC B cells remodel their immunoglobulin (Ig) genes by somatic hypermutation (SHM) and class switch recombination (CSR) in their Ig genes. Activation Induced Deaminase (AID) initiates CSR and SHM by generating U:G mismatches on Ig DNA that can then be processed by Uracyl-N-glycosylase (UNG). AID promotes collateral damage in the form of chromosome translocations and off-target SHM, however, the exact contribution of AID activity to lymphoma generation and progression is not completely understood. Here we show using a conditional knock-in strategy that AID supra-activity alone is not sufficient to generate B cell transformation. In contrast, in the absence of UNG, AID supra-expression increases SHM and promotes lymphoma. Whole exome sequencing revealed that AID heavily contributes to lymphoma SHM, promoting subclonal variability and a wider range of oncogenic variants. Thus, our data provide direct evidence that UNG is a brake to AID-induced intratumoral heterogeneity and evolution of B cell lymphoma.


Assuntos
Citidina Desaminase/genética , Heterogeneidade Genética , Linfoma de Células B/genética , Uracila-DNA Glicosidase/genética , Animais , Transformação Celular Neoplásica/genética , Células Cultivadas , Evolução Clonal , Citidina Desaminase/metabolismo , Feminino , Linfoma de Células B/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Mutação , Uracila-DNA Glicosidase/metabolismo
SELEÇÃO DE REFERÊNCIAS
DETALHE DA PESQUISA
...