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1.
Anal Chim Acta ; 1175: 338741, 2021 Aug 29.
Artigo em Inglês | MEDLINE | ID: mdl-34330449

RESUMO

8-oxoguanine DNA glycosylase (OGG), which plays a crucial role in base excision repair (BER), is an important biomarker. The existing highly sensitive fluorescent methods always need complicated amplification design. The method with high sensitivity and simple design at the same time is urgently needed. Here, we developed a highly sensitive detection method for OGG detection with lambda exonuclease and the background signal suppression probe. Through probe structure design, the steric hindrance and competitive binding effects successfully suppressed the background signal. We achieved sensitive detection of OGG with a simple design, and the limit of detection was 5.0 × 10-4 U mL-1. Moreover, the method was highly selective and successfully applied to OGG detection in biological samples, which shows the potential clinical application value.


Assuntos
DNA Glicosilases , DNA Glicosilases/metabolismo , Reparo do DNA , Guanina/análogos & derivados
2.
Nat Commun ; 12(1): 4108, 2021 07 05.
Artigo em Inglês | MEDLINE | ID: mdl-34226550

RESUMO

DNA glycosylases must distinguish the sparse damaged sites from the vast expanse of normal DNA bases. However, our understanding of the nature of nucleobase interrogation is still limited. Here, we show that hNEIL1 (human endonuclease VIII-like 1) captures base lesions via two competing states of interaction: an activated state that commits catalysis and base excision repair, and a quarantine state that temporarily separates and protects the flipped base via auto-inhibition. The relative dominance of the two states depends on key residues of hNEIL1 and chemical properties (e.g. aromaticity and hydrophilicity) of flipped bases. Such a DNA repair mechanism allows hNEIL1 to recognize a broad spectrum of DNA damage while keeps potential gratuitous repair in check. We further reveal the molecular basis of hNEIL1 activity regulation mediated by post-transcriptional modifications and provide an example of how exquisite structural dynamics serves for orchestrated enzyme functions.


Assuntos
DNA Glicosilases/química , DNA Glicosilases/metabolismo , Reparo do DNA/fisiologia , Triagem , Sequência de Aminoácidos , Sítios de Ligação , Domínio Catalítico , DNA/química , Dano ao DNA , DNA Glicosilases/genética , Desoxirribonuclease (Dímero de Pirimidina)/química , Desoxirribonuclease (Dímero de Pirimidina)/genética , Desoxirribonuclease (Dímero de Pirimidina)/metabolismo , Humanos , Simulação de Dinâmica Molecular , Mutação , Conformação Proteica , Especificidade por Substrato
3.
Se Pu ; 39(7): 721-729, 2021 Jul 08.
Artigo em Chinês | MEDLINE | ID: mdl-34227370

RESUMO

8-Oxoguanine DNA glycosylase (OGG1) is an important enzyme that plays a key role in oxidative DNA damage repair. OGG1 can specifically recognize and excise 8-oxoG (a product of oxidative damage found in double-stranded DNA) through base excision repair (BER). OGG1 is expressed in normal tissues, and in most tumor tissues. Oxidative cellular damage can produce an inflammatory reaction, alleviating some measure of constitutive OGG1 inhibition. OGG1 inhibition in cancer cells shows some promise as a new method of cancer treatment. Most current OGG1 research focuses on regulating OGG1 with targeted small molecules. To date, no aptamer screen for OGG1 has been reported. Aptamers are single-stranded DNA (ssDNA) or RNA oligonucleotides that can bind to a target with high affinity and specificity in vitro, that can be identified by systematic evolution of ligands by exponential enrichment (SELEX). Aptamers can be used as chemical ligands to regulate intermolecular interactions. In this study, a screen for aptamers with OGG1 affinity was performed for the first time. Capillary electrophoresis (CE) is a microanalytical technique that offers speed and high separation efficiency. In this work, two screening methods based on CE-SELEX technology were established: a one-round pressure controllable selection, and a multi-round selection. The most important criterion for successful one-round pressure controllable selection is to select a competitive target with a different CE migration time than that of the target of interest. We mixed OGG1 with a competitive target and a nucleic acid library for CE analysis. Two proteins competitively bind sequences in the library, forming independent complexes. The concentration of the competitive target is continuously increased until complexes with the target stop decreasing, indicating that the target and the ssDNA library have formed a stable complex. Complexes were collected for PCR amplification, purification, and high-throughput sequencing to obtain high affinity aptamers. This method greatly improves screening efficiency, and reduces non-specific binding to the target, which is helpful for obtaining aptamers with high affinity and specificity. One-round pressure controllable selection for high affinity OGG1 selective aptamers was performed using single strand binding protein (SSB) to competitively and tightly bind nucleic acids in the library. The competitive screening pressure was increased by increasing the SSB concentration to eliminate sequences with low affinity for OGG1 from the random oligonucleotide library. Nucleic acid sequences with high OGG1 affinity were obtainable in one step, and OGG1-ssDNA complexes were collected by creating a timed program on Beckman P/ACE MDQ capillary electrophoresis. Collection occurred from 2.2 to 2.8 min. Under identical incubation and electrophoresis conditions, multiple round selections were conducted by injecting samples of co-incubated nucleic acid library and target into the capillary. After separation under a high-voltage electric field, nucleic acid target complexes were collected, amplified by PCR, purified, and used as an enriched secondary library in the next round of screening. High affinity aptamers were generally obtained within three rounds. Comparing results of the two screening methods, the three candidate aptamer sequences found with the highest frequency were consistent, and displayed KD values ranging from 1.71 to 2.64 µmol/L. Molecular docking analysis suggests that Apt 1 may bind to the OGG1 active pocket, which functions to repair oxidative damage. Comparison of the two screening methods indicates that one-round pressure controllable selection is more rapid and efficient, providing guidance for the design of other protein aptamer screening methods. The obtained aptamer is expected to be function effectively as an OGG1-mediated DNA repair inhibitor.


Assuntos
Aptâmeros de Nucleotídeos , DNA Glicosilases , Aptâmeros de Nucleotídeos/química , DNA Glicosilases/química , DNA de Cadeia Simples , Eletroforese Capilar , Guanina/análogos & derivados , Simulação de Acoplamento Molecular , Técnica de Seleção de Aptâmeros
4.
Int J Colorectal Dis ; 36(10): 2199-2204, 2021 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-34244858

RESUMO

PURPOSE: The carrier frequency of MUTYH pathogenic variants in the population may be as high as one in 45. Some studies have found an increased risk of colorectal cancer (CRC) in monoallelic carriers of MUTYH pathogenic variants, but the role of early surveillance colonoscopy is not conclusive. This study aimed to assess the outcomes of colonoscopy surveillance in MUTYH carriers. METHODS: Patients, with a monoallelic pathogenic variant in MUTYH, found at cascade testing, were identified from the St Mark's Hospital Polyposis Registry database. Findings at surveillance colonoscopy were reviewed. RESULTS: Two hundred and forty-nine carriers were identified, of whom 125 had undergone at least one surveillance colonoscopy. Twenty-eight patients (22%) developed at least one adenoma; all adenomas had low-grade dysplasia (LGD). The median age at first colonoscopy was 36 years (range 16-75 years). The median age at first adenoma detection was 43 years (range 22-75 years). The cumulative incidence of adenoma development by age 30, 40, 50, 60 and 70 years was 3.2%, 8.8%, 15.2%, 18.4% and 20.8%, respectively. No CRCs were observed. CONCLUSIONS: Our cohort of monoallelic carriers of MUTYH pathogenic variants is a relatively younger group than adults entering population screening colonoscopy, but a high adenoma rate was not observed. No CRCs were detected, suggesting that current guidance that these individuals should be managed in the same way as the general population is reasonable.


Assuntos
Adenoma , Polipose Adenomatosa do Colo , Neoplasias Colorretais , DNA Glicosilases , Adenoma/epidemiologia , Adenoma/genética , Adolescente , Adulto , Idoso , Neoplasias Colorretais/genética , DNA Glicosilases/genética , Heterozigoto , Humanos , Pessoa de Meia-Idade , Mutação , Adulto Jovem
5.
Commun Biol ; 4(1): 876, 2021 07 15.
Artigo em Inglês | MEDLINE | ID: mdl-34267321

RESUMO

The multi-step base excision repair (BER) pathway is initiated by a set of enzymes, known as DNA glycosylases, able to scan DNA and detect modified bases among a vast number of normal bases. While DNA glycosylases in the BER pathway generally bend the DNA and flip damaged bases into lesion specific pockets, the HEAT-like repeat DNA glycosylase AlkD detects and excises bases without sequestering the base from the DNA helix. We show by single-molecule tracking experiments that AlkD scans DNA without forming a stable interrogation complex. This contrasts with previously studied repair enzymes that need to flip bases into lesion-recognition pockets and form stable interrogation complexes. Moreover, we show by design of a loss-of-function mutant that the bimodality in scanning observed for the structural homologue AlkF is due to a key structural differentiator between AlkD and AlkF; a positively charged ß-hairpin able to protrude into the major groove of DNA.


Assuntos
Proteínas de Bactérias/genética , DNA Glicosilases/genética , DNA Bacteriano/genética , Proteínas de Bactérias/metabolismo , DNA Glicosilases/metabolismo
6.
Cells ; 10(7)2021 06 24.
Artigo em Inglês | MEDLINE | ID: mdl-34202661

RESUMO

It was proposed that the last universal common ancestor (LUCA) evolved under high temperatures in an oxygen-free environment, similar to those found in deep-sea vents and on volcanic slopes. Therefore, spontaneous DNA decay, such as base loss and cytosine deamination, was the major factor affecting LUCA's genome integrity. Cosmic radiation due to Earth's weak magnetic field and alkylating metabolic radicals added to these threats. Here, we propose that ancient forms of life had only two distinct repair mechanisms: versatile apurinic/apyrimidinic (AP) endonucleases to cope with both AP sites and deaminated residues, and enzymes catalyzing the direct reversal of UV and alkylation damage. The absence of uracil-DNA N-glycosylases in some Archaea, together with the presence of an AP endonuclease, which can cleave uracil-containing DNA, suggests that the AP endonuclease-initiated nucleotide incision repair (NIR) pathway evolved independently from DNA glycosylase-mediated base excision repair. NIR may be a relic that appeared in an early thermophilic ancestor to counteract spontaneous DNA damage. We hypothesize that a rise in the oxygen level in the Earth's atmosphere ~2 Ga triggered the narrow specialization of AP endonucleases and DNA glycosylases to cope efficiently with a widened array of oxidative base damage and complex DNA lesions.


Assuntos
DNA Glicosilases/metabolismo , Reparo do DNA , Evolução Molecular , Oxigênio/metabolismo , Alquilação , Animais , Dano ao DNA , Humanos
7.
Nucleic Acids Res ; 49(14): 8177-8188, 2021 08 20.
Artigo em Inglês | MEDLINE | ID: mdl-34232996

RESUMO

The oxidative base damage, 8-oxo-7,8-dihydroguanine (8-oxoG) is a highly mutagenic lesion because replicative DNA polymerases insert adenine (A) opposite 8-oxoG. In mammalian cells, the removal of A incorporated across from 8-oxoG is mediated by the glycosylase MUTYH during base excision repair (BER). After A excision, MUTYH binds avidly to the abasic site and is thus product inhibited. We have previously reported that UV-DDB plays a non-canonical role in BER during the removal of 8-oxoG by 8-oxoG glycosylase, OGG1 and presented preliminary data that UV-DDB can also increase MUTYH activity. In this present study we examine the mechanism of how UV-DDB stimulates MUTYH. Bulk kinetic assays show that UV-DDB can stimulate the turnover rate of MUTYH excision of A across from 8-oxoG by 4-5-fold. Electrophoretic mobility shift assays and atomic force microscopy suggest transient complex formation between MUTYH and UV-DDB, which displaces MUTYH from abasic sites. Using single molecule fluorescence analysis of MUTYH bound to abasic sites, we show that UV-DDB interacts directly with MUTYH and increases the mobility and dissociation rate of MUTYH. UV-DDB decreases MUTYH half-life on abasic sites in DNA from 8800 to 590 seconds. Together these data suggest that UV-DDB facilitates productive turnover of MUTYH at abasic sites during 8-oxoG:A repair.


Assuntos
Dano ao DNA/efeitos dos fármacos , DNA Glicosilases/genética , Guanina/análogos & derivados , Estresse Oxidativo/efeitos dos fármacos , Adenina/química , Animais , Dano ao DNA/efeitos da radiação , Reparo do DNA/efeitos dos fármacos , Reparo do DNA/efeitos da radiação , Replicação do DNA/efeitos dos fármacos , Replicação do DNA/efeitos da radiação , Guanina/química , Guanina/farmacologia , Guanina/toxicidade , Hidrocarbonetos Clorados/farmacologia , Hidrocarbonetos Clorados/toxicidade , Camundongos , Estresse Oxidativo/efeitos da radiação , Imagem Individual de Molécula
8.
Nucleic Acids Res ; 49(12): 7154-7163, 2021 07 09.
Artigo em Inglês | MEDLINE | ID: mdl-34142156

RESUMO

Mammalian MutY homologue (MUTYH) is an adenine DNA glycosylase that excises adenine inserted opposite 8-oxoguanine (8-oxoG). The inherited variations in human MUTYH gene are known to cause MUTYH-associated polyposis (MAP), which is associated with colorectal cancer. MUTYH is involved in base excision repair (BER) with proliferating cell nuclear antigen (PCNA) in DNA replication, which is unique and critical for effective mutation-avoidance. It is also reported that MUTYH has a Zn-binding motif in a unique interdomain connector (IDC) region, which interacts with Rad9-Rad1-Hus1 complex (9-1-1) in DNA damage response, and with apurinic/apyrimidinic endonuclease 1 (APE1) in BER. However, the structural basis for the BER pathway by MUTYH and its interacting proteins is unclear. Here, we determined the crystal structures of complexes between mouse MUTYH and DNA, and between the C-terminal domain of mouse MUTYH and human PCNA. The structures elucidated the repair mechanism for the A:8-oxoG mispair including DNA replication-coupled repair process involving MUTYH and PCNA. The Zn-binding motif was revealed to comprise one histidine and three cysteine residues. The IDC, including the Zn-binding motif, is exposed on the MUTYH surface, suggesting its interaction modes with 9-1-1 and APE1, respectively. The structure of MUTYH explains how MAP mutations perturb MUTYH function.


Assuntos
DNA Glicosilases/química , Adenina , Polipose Adenomatosa do Colo/genética , Motivos de Aminoácidos , Animais , DNA/química , DNA Glicosilases/genética , Reparo do DNA , Replicação do DNA , Guanina/análogos & derivados , Humanos , Camundongos , Modelos Moleculares , Mutação , Antígeno Nuclear de Célula em Proliferação/química , Zinco
9.
DNA Repair (Amst) ; 105: 103160, 2021 09.
Artigo em Inglês | MEDLINE | ID: mdl-34192601

RESUMO

GO system is part of base excision DNA repair and is required for the correct repair of 8-oxoguanine (8-oxoG), one of the most abundant oxidative lesions. Due to the ability of 8-oxoG to mispair with A, this base is highly mutagenic, and its repair requires two enzymes: Fpg that removes 8-oxoG from 8-oxoG:C pairs, and MutY that excises the normal A from 8-oxoG:A mispairs. Here we characterize the properties of putative GO system DNA glycosylases from Staphylococcus aureus, an important human opportunistic pathogen that causes hospital infections and presents a serious health concern due to quick spread of antibiotic-resistant strains. In addition to Fpg and MutY from the reference NCTC 8325 strain (SauFpg1 and SauMutY), we have also studied an Fpg homolog from a multidrug-resistant C0673 isolate (SauFpg2), which is different from SauFpg1 in its sequence. Both SauFpg enzymes showed the highest activity at pH 7.0-9.0 and NaCl concentrations 25-75 mM (SauFpg1) or 50-100 mM (SauFpg2), whereas SauMutY was active at a broad pH range and had a salt optimum at ∼75 mM NaCl. Both SauFpg1 and SauFpg2 bound and cleaved duplexes containing 8-oxoG, 5-hydroxyuracil, 5,6-dihydrouracil or apurinic/apyrimidinic site paired with C, T, or G, but not with A. For SauFpg1 and SauFpg2, 8-oxoG was the best substrate tested, and 5,6-dihydrouracil was the worst one. SauMutY efficiently excised adenine from duplex substrates containing A:8-oxoG or A:G pairs. SauFpg enzymes were readily trapped on DNA by NaBH4 treatment, indicating formation of a Schiff base reaction intermediate. Surprisingly, SauMutY was also trapped significantly better than its E. coli homolog. All three S. aureus GO glycosylases drastically reduced spontaneous mutagenesis when expressed in an fpg mutY E. coli double mutant. Overall, we conclude that S. aureus possesses an active GO system, which could possibly be targeted for sensitization of this pathogen to oxidative stress.


Assuntos
Dano ao DNA , DNA Glicosilases/metabolismo , DNA-Formamidopirimidina Glicosilase/metabolismo , Guanina/análogos & derivados , Staphylococcus aureus/enzimologia , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Sequência de Bases , DNA Glicosilases/genética , DNA Bacteriano/metabolismo , DNA-Formamidopirimidina Glicosilase/genética , Guanina/metabolismo , Concentração de Íons de Hidrogênio , Filogenia , Alinhamento de Sequência , Staphylococcus aureus/genética , Staphylococcus aureus/metabolismo , Especificidade por Substrato
10.
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
11.
DNA Repair (Amst) ; 105: 103152, 2021 09.
Artigo em Inglês | MEDLINE | ID: mdl-34186496

RESUMO

The Polb gene encodes DNA polymerase beta (Pol ß), a DNA polymerase that functions in base excision repair (BER) and microhomology-mediated end-joining. The Pol ß-Y265C protein exhibits low catalytic activity and fidelity, and is also deficient in microhomology-mediated end-joining. We have previously shown that the PolbY265C/+ and PolbY265C/C mice develop lupus. These mice exhibit high levels of antinuclear antibodies and severe glomerulonephritis. We also demonstrated that the low catalytic activity of the Pol ß-Y265C protein resulted in accumulation of BER intermediates that lead to cell death. Debris released from dying cells in our mice could drive development of lupus. We hypothesized that deletion of the Neil1 and Ogg1 DNA glycosylases that act upstream of Pol ß during BER would result in accumulation of fewer BER intermediates, resulting in less severe lupus. We found that high levels of antinuclear antibodies are present in the sera of PolbY265C/+ mice deleted of Ogg1 and Neil1 DNA glycosylases. However, these mice develop significantly less severe renal disease, most likely due to high levels of IgM in their sera.


Assuntos
DNA Glicosilases/metabolismo , DNA Polimerase beta/metabolismo , Reparo do DNA , Lúpus Eritematoso Sistêmico/enzimologia , Mutação , Estresse Oxidativo , Animais , DNA/metabolismo , DNA Glicosilases/genética , DNA Polimerase beta/genética , Modelos Animais de Doenças , Deleção de Genes , Lúpus Eritematoso Sistêmico/genética , Lúpus Eritematoso Sistêmico/metabolismo , Camundongos
12.
Biochem J ; 478(12): 2359-2370, 2021 06 25.
Artigo em Inglês | MEDLINE | ID: mdl-34060590

RESUMO

The oxidatively generated genotoxic spiroiminodihydantoin (Sp) lesions are well-known substrates of the base excision repair (BER) pathway initiated by the bifunctional DNA glycosylase NEIL1. In this work, we reported that the excision kinetics of the single Sp lesions site-specifically embedded in the covalently closed circular DNA plasmids (contour length 2686 base pairs) by NEIL1 are biphasic under single-turnover conditions ([NEIL1] ≫ [SpDNApl]) in contrast with monophasic excision kinetics of the same lesions embedded in147-mer Sp-modified DNA duplexes. Under conditions of a large excess of plasmid DNA base pairs over NEIL1 molecules, the kinetics of excision of Sp lesions are biphasic in nature, exhibiting an initial burst phase, followed by a slower rate of formation of excision products The burst phase is associated with NEIL1-DNA plasmid complexes, while the slow kinetic phase is attributed to the dissociation of non-specific NEIL1-DNA complexes. The amplitude of the burst phase is limited because of the competing non-specific binding of NEIL1 to unmodified DNA sequences flanking the lesion. A numerical analysis of the incision kinetics yielded a value of φ ≍ 0.03 for the fraction of NEIL1 encounters with plasmid molecules that result in the excision of the Sp lesion, and a characteristic dissociation time of non-specific NEIL1-DNA complexes (τ-ns ≍ 8 s). The estimated average DNA translocation distance of NEIL1 is ∼80 base pairs. This estimate suggests that facilitated diffusion enhances the probability that NEIL1 can locate its substrate embedded in an excess of unmodified plasmid DNA nucleotides by a factor of ∼10.


Assuntos
Dano ao DNA , DNA Glicosilases/metabolismo , Reparo do DNA , DNA/química , DNA Glicosilases/genética , Células HeLa , Humanos , Oxirredução , Plasmídeos/genética
13.
J Biol Chem ; 296: 100723, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-33932404

RESUMO

Aberrant or constitutive activation of nuclear factor kappa B (NF-κB) contributes to various human inflammatory diseases and malignancies via the upregulation of genes involved in cell proliferation, survival, angiogenesis, inflammation, and metastasis. Thus, inhibition of NF-κB signaling has potential for therapeutic applications in cancer and inflammatory diseases. We reported previously that Nei-like DNA glycosylase 2 (NEIL2), a mammalian DNA glycosylase, is involved in the preferential repair of oxidized DNA bases from the transcriptionally active sequences via the transcription-coupled base excision repair pathway. We have further shown that Neil2-null mice are highly sensitive to tumor necrosis factor α (TNFα)- and lipopolysaccharide-induced inflammation. Both TNFα and lipopolysaccharide are potent activators of NF-κB. However, the underlying mechanism of NEIL2's role in the NF-κB-mediated inflammation remains elusive. Here, we have documented a noncanonical function of NEIL2 and demonstrated that the expression of genes, such as Cxcl1, Cxcl2, Cxcl10, Il6, and Tnfα, involved in inflammation and immune cell migration was significantly higher in both mock- and TNFα-treated Neil2-null mice compared with that in the WT mice. NEIL2 blocks NF-κB's binding to target gene promoters by directly interacting with the Rel homology region of RelA and represses proinflammatory gene expression as determined by co-immunoprecipitation, chromatin immunoprecipitation, and electrophoretic mobility-shift assays. Remarkably, intrapulmonary administration of purified NEIL2 via a noninvasive nasal route significantly abrogated binding of NF-κB to cognate DNA, leading to decreased expression of proinflammatory genes and neutrophil recruitment in Neil2-null as well as WT mouse lungs. Our findings thus highlight the potential of NEIL2 as a biologic for inflammation-associated human diseases.


Assuntos
DNA Glicosilases/metabolismo , Pulmão/metabolismo , NF-kappa B/metabolismo , Animais , Movimento Celular , Regulação da Expressão Gênica , Inflamação/metabolismo , Pulmão/patologia , Camundongos , Transdução de Sinais
14.
J Chem Theory Comput ; 17(6): 3525-3538, 2021 Jun 08.
Artigo em Inglês | MEDLINE | ID: mdl-34018392

RESUMO

The recognition mechanism of oxidative damage in organisms has long been a research hotspot. Water is an important medium in the recognition process, but its specific role remains unknown. There is a need to develop a suitable force field that can adequately describe the electrostatic, hydrogen bond, and other interactions among the molecules in the complex system of the repair enzyme and oxidized base. The developing ABEEM polarizable force field (PFF) has been used to simulate the repaired enzyme hOGG1 and oxidized DNA (PDB ID: 1EBM) in a biological environment, and the corresponding results are better than those of the fixed-charge force fields OPLS/AA and AMBER OL15. 8-Oxo-G is recognized by Gln315 of hOGG1 mainly through hydrogen bonds mediated by continuous exchange of 2 water molecules. Phe319 and Cys253 are stacked on both sides of the π planes of bases to form sandwich structures. The charge polarization effect gives an important signal to drive the exchange of water molecules and maintains the recognition of oxidation bases by enzymes. The mediated main water molecule A and mediated auxiliary water molecule B together pull Gln315 to recognize 8-oxo-G by hydrogen bond interactions. Then, the charge polarization signal of solvent water molecule C with a large absolute charge causes the absolute charge of O atoms in water molecule A or B to increase by approximately 0.2 e, and water molecule A or B leaves Gln315 and 8-oxo-G. The other water molecule and water molecule C synergistically recognize 8-oxo-G with Gln315. Even though the water molecules between Gln315 and 8-oxo-G are removed, the MD simulation results show that water molecules appear between Gln315 and 8-oxo-G in a very short time (<2 ps). The dwell time of each water molecule is approximately 60 ps. The radial distribution function and dwell time support the correctness of the above mechanism. These polarization effects and hydrogen bonding interactions cannot be simulated by a fixed-charge force field.


Assuntos
DNA Glicosilases/metabolismo , Guanina/metabolismo , Água/metabolismo , 8-Hidroxi-2'-Desoxiguanosina/química , 8-Hidroxi-2'-Desoxiguanosina/metabolismo , DNA/química , DNA/metabolismo , DNA Glicosilases/química , Guanina/química , Humanos , Ligação de Hidrogênio , Simulação de Dinâmica Molecular , Oxirredução , Teoria Quântica , Água/química
15.
Food Chem ; 361: 130102, 2021 Nov 01.
Artigo em Inglês | MEDLINE | ID: mdl-34029891

RESUMO

Protein glycation and formation of advanced glycation end-products (AGEs) impose threats to the human health. This study firstly investigated the inhibition of Chinese bayberry (Myrica rubra) phenolics on AGEs formation through mechanistic analysis. Four common Chinese bayberry cultivars were selected to prepare phenolic-rich extracts (CBEs) and characterized for phenolic composition, and their anti-AGE properties were evaluated in multiple in vitro systems. Total sixteen phenolics were quantified in CBEs by UPLC-ESI-MS/MS. CBEs reduced total and specific fluorescent AGEs formation in various simulating models, and protected the protein from structural modification, oxidation, and cross-linking. Mechanistic analysis unveiled that scavenging of free radicals, inactivation of transition metals, interaction with protein to form complexes, and trapping of reactive α-dicarbonyls to form adducts underlain the mechanisms of the anti-glycative actions of CBEs. Chinese bayberry fruits, especially the cultivars Biqi and Wuzi, may be a promising dietary strategy to mitigate AGEs load in the human body.


Assuntos
Produtos Finais de Glicação Avançada/metabolismo , Myrica/química , Fenóis/farmacologia , DNA Glicosilases/efeitos dos fármacos , Frutas/química , Oxirredução/efeitos dos fármacos , Fenóis/análise
16.
Breast Cancer Res Treat ; 188(1): 295-305, 2021 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-33942220

RESUMO

BACKGROUND: Breast cancer (BC) is the most common cancer in women. In contrast, male BC is about 100 times less common than in women, being considered a rare disease. Male BC may be a distinctive subtype of BC and available data seems to indicate that male BC has a higher dependence on genetic variants than female BC. Nevertheless, the same prognostic and predictive markers are used to determine optimal management strategies for both male and female BC. Several studies have assessed the role of genetic polymorphisms (SNPs) in DNA repair genes in female BC susceptibility. However, data on male BC is scarce. Thus, the current study aimed to assess the role of SNPs in XRCC1, MUTYH and TP53 genes in a male cohort of BC, and, in addition, compare the male data with matched results previously genotyped in female BC patients. METHODS: The male BC cohort was genotyped through Real-Time PCR using TaqMan Assays for several SNPs previously analysed in Portuguese female BC patients. RESULTS: The results obtained indicate significant differences in BC susceptibility between males and females for the XRCC1 rs1799782, MUTYH rs3219489 and TP53 rs1042522 and rs8064946 variants. CONCLUSIONS: In males, XRCC1 and TP53 variants, when in heterozygosity, seem to be related with lower susceptibility for BC, contrasting with higher susceptibility for a MUTYH variant in females. These findings may help to explain the difference in incidence of BC between the two sexes.


Assuntos
Neoplasias da Mama Masculina/genética , DNA Glicosilases/genética , Proteína Supressora de Tumor p53/genética , Proteína 1 Complementadora Cruzada de Reparo de Raio-X/genética , Neoplasias da Mama , Estudos de Casos e Controles , Estudos de Coortes , Feminino , Predisposição Genética para Doença , Genótipo , Humanos , Masculino , Polimorfismo de Nucleotídeo Único
17.
Redox Biol ; 43: 101997, 2021 07.
Artigo em Inglês | MEDLINE | ID: mdl-33965877

RESUMO

The oxidation-induced DNA modification 8-oxo-7,8-dihydro-2'-deoxyguanosine (8-oxodG) was recently implicated in the activation and repression of gene transcription. We aimed at a systematic characterisation of the impacts of 8-oxodG on the activity of a GC box placed upstream from the RNA polymerase II core promoter. With the help of reporters carrying single synthetic 8-oxodG residues at four conserved G:C base pairs (underlined) within the 5'-TGGGCGGAGC-3' GC box sequence, we identified two modes of interference of 8-oxodG with the promoter activity. Firstly, 8-oxodG in the purine-rich (but not in the pyrimidine-rich) strand caused direct impairment of transcriptional activation. In addition, and independently of the first mechanism, 8-oxodG initiated a decline of the gene expression, which was mediated by the specific DNA glycosylase OGG1. For the different 8-oxodG positions, the magnitude of this effect reflected the excision preferences of OGG1. Thus, 8-oxodG seeded in the pyrimidine-rich strand was excised with the highest efficiency and caused the most pronounced decrease of the promoter activity. Conversely, 8-oxodG in the symmetric position within the same CpG dinucleotide, was poorly excised and induced no decline of the gene expression. Of note, abasic lesions caused gene silencing in both positions. By contrast, an uncleavable apurinic lesion in the pyrimidine-rich strand enhanced the GC box activity, suggesting that the AP endonuclease step provides a switch between the active versus repressed promoter states during base excision repair.


Assuntos
DNA Glicosilases , Reparo do DNA , Dano ao DNA , DNA Glicosilases/genética , DNA Liase (Sítios Apurínicos ou Apirimidínicos) , Guanina/análogos & derivados
18.
Clin Transl Gastroenterol ; 12(5): e00353, 2021 05 17.
Artigo em Inglês | MEDLINE | ID: mdl-33999013

RESUMO

INTRODUCTION: Familial adenomatous polyposis (FAP) is a hereditary colorectal cancer (CRC) syndrome characterized by accelerated adenoma development due to inherited (or de novo) mutations in the APC regulator of WNT signaling pathway (APC) gene. The mechanism underlying this accelerated polyp development in subjects with FAP has not been defined. Given that LGR5+ stem cells drive crypt cell proliferation, we hypothesized that FAP crypts would demonstrate aberrant leucine-rich repeat-containing G-protein-coupled receptor 5 (LGR5) staining patterns. METHODS: Biopsies were taken from 11 healthy subjects, 7 subjects with Lynch syndrome, 4 subjects with FAP, and 1 subject with MUTYH-associated polyposis syndrome during routine screening or surveillance colonoscopy. Crypt staining was evaluated by immunohistochemistry of paraffin-embedded tissue sections. Stem cell numbers were estimated by immunofluorescence staining of isolated crypts using antibodies against LGR5 and other proteins. RESULTS: Subjects with FAP exhibited a greater number of LGR5+ stem cells in their crypts than healthy subjects and subjects with Lynch syndrome and MUTYH-associated polyposis syndrome. Most crypts of subjects with FAP harbored LGR5+ cells located above the lower third of the crypts. DISCUSSION: These findings support a model in which inactivation of one copy of APC leads to increased numbers of LGR5+ stem cells, many of which are ectopic, in colon crypts of subjects with FAP. Overabundant and ectopic LGR5+ stem cells could lead to an expanded proliferative zone of dividing cells more likely to develop mutations that would contribute to the accelerated adenoma development observed in FAP.


Assuntos
Polipose Adenomatosa do Colo/patologia , Colo/patologia , Receptores Acoplados a Proteínas G/análise , Células-Tronco/patologia , Adulto , Idoso , Biópsia , Estudos de Casos e Controles , Proliferação de Células , Neoplasias Colorretais Hereditárias sem Polipose/patologia , DNA Glicosilases/análise , Feminino , Imunofluorescência , Humanos , Imuno-Histoquímica , Masculino , Microscopia Confocal , Pessoa de Meia-Idade , Adulto Jovem
19.
Genes (Basel) ; 12(3)2021 03 22.
Artigo em Inglês | MEDLINE | ID: mdl-33809907

RESUMO

There is a paucity of data on how gene expression enables identification of individuals who are at risk of exposure to carcinogens from e-cigarette (e-cig) vaping; and how human vaping behaviors modify these exposures. This pilot study aimed to identify genes regulated from acute exposure to e-cig using RT-qPCR. Three subjects (2M and 1F) made three visits to the lab (nTOT = 9 visits); buccal and blood samples were collected before and immediately after scripted vaping 20 puffs (nTOT = 18 samples); vaping topography data were collected in each session. Subjects used their own e-cig containing 50:50 propylene glycol (PG):vegetable glycerine (VG) +3-6 mg/mL nicotine. The tumor suppressor TP53 was significantly upregulated in buccal samples. TP53 expression was puff volume and flow rate dependent in both tissues. In blood, the significant downregulation of N-methylpurine DNA glycosylase (MPG), a base excision repair gene, was consistent across all subjects. In addition to DNA repair pathway, cell cycle and cancer pathways were the most enriched pathways in buccal and blood samples, respectively. This pilot study demonstrates that vaping 20 puffs significantly alters expression of TP53 in human tissues; vaping behavior is an important modifier of this response. A larger study is needed to confirm these relationships.


Assuntos
Dano ao DNA , DNA Glicosilases/sangue , Neoplasias/genética , Nicotina/efeitos adversos , Proteína Supressora de Tumor p53/genética , DNA Glicosilases/genética , Regulação para Baixo , Sistemas Eletrônicos de Liberação de Nicotina , Estudos de Viabilidade , Feminino , Regulação da Expressão Gênica/efeitos dos fármacos , Humanos , Masculino , Boca/química , Projetos Piloto , Regulação para Cima
20.
PLoS Pathog ; 17(4): e1009502, 2021 04.
Artigo em Inglês | MEDLINE | ID: mdl-33826673

RESUMO

Trypanosoma cruzi is the etiologic agent of Chagas' disease. Infected cells with T. cruzi activate several responses that promote unbalance of reactive oxygen species (ROS) that may cause DNA damage that activate cellular responses including DNA repair processes. In this work, HeLa cells and AC16 human cardiomyocyte cell line were infected with T. cruzi to investigate host cell responses at genome level during parasites intracellular life cycle. In fact, alkaline sensitive sites and oxidized DNA bases were detected in the host cell genetic material particularly in early stages of infection. These DNA lesions were accompanied by phosphorylation of the histone H2Ax, inducing γH2Ax, a marker of genotoxic stress. Moreover, Poly [ADP-ribose] polymerase-1 (PARP1) and 8-oxoguanine glycosylase (OGG1) are recruited to host cell nuclei, indicating activation of the DNA repair process. In infected cells, chromatin-associated proteins are carbonylated, as a possible consequence of oxidative stress and the nuclear factor erythroid 2-related factor 2 (NRF2) is induced early after infection, suggesting that the host cell antioxidant defenses are activated. However, at late stages of infection, NRF2 is downregulated. Interestingly, host cells treated with glutathione precursor, N-acetyl cysteine, NRF2 activator (Sulforaphane), and also Benznidonazol (BNZ) reduce parasite burst significantly, and DNA damage. These data indicate that the balance of oxidative stress and DNA damage induction in host cells may play a role during the process of infection itself, and interference in these processes may hamper T. cruzi infection, revealing potential target pathways for the therapy support.


Assuntos
Doença de Chagas/parasitologia , Dano ao DNA , Interações Hospedeiro-Parasita , Estresse Oxidativo , Trypanosoma cruzi/fisiologia , Antioxidantes/metabolismo , Morte Celular , Linhagem Celular , DNA Glicosilases/genética , DNA Glicosilases/metabolismo , Reparo do DNA , Regulação para Baixo , Células HeLa , Histonas/genética , Histonas/metabolismo , Humanos , Fator 2 Relacionado a NF-E2/genética , Fator 2 Relacionado a NF-E2/metabolismo , Fosforilação , Poli(ADP-Ribose) Polimerase-1/genética , Poli(ADP-Ribose) Polimerase-1/metabolismo , Espécies Reativas de Oxigênio/metabolismo , Trypanosoma cruzi/patogenicidade
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