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1.
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
2.
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
3.
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
4.
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
5.
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
6.
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
7.
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
8.
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
9.
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
10.
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
11.
Int J Mol Sci ; 22(9)2021 Apr 27.
Artigo em Inglês | MEDLINE | ID: mdl-33925271

RESUMO

DNA damage caused by reactive oxygen species may result in genetic mutations or cell death. Base excision repair (BER) is the major pathway that repairs DNA oxidative damage in order to maintain genomic integrity. In mammals, eleven DNA glycosylases have been reported to initiate BER, where each recognizes a few related DNA substrate lesions with some degree of overlapping specificity. 7,8-dihydro-8-oxoguanine (8-oxoG), one of the most abundant DNA oxidative lesions, is recognized and excised mainly by 8-oxoguanine DNA glycosylase 1 (OGG1). Further oxidation of 8-oxoG generates hydantoin lesions, which are recognized by NEIL glycosylases. Here, we demonstrate that NEIL1, and to a lesser extent NEIL2, can potentially function as backup BER enzymes for OGG1 upon pharmacological inhibition or depletion of OGG1. NEIL1 recruitment kinetics and chromatin binding after DNA damage induction increase in cells treated with OGG1 inhibitor TH5487 in a dose-dependent manner, whereas NEIL2 accumulation at DNA damage sites is prolonged following OGG1 inhibition. Furthermore, depletion of OGG1 results in increased retention of NEIL1 and NEIL2 at damaged chromatin. Importantly, oxidatively stressed NEIL1- or NEIL2-depleted cells show excessive genomic 8-oxoG lesions accumulation upon OGG1 inhibition, suggesting a prospective compensatory role for NEIL1 and NEIL2. Our study thus exemplifies possible backup mechanisms within the base excision repair pathway.


Assuntos
DNA Glicosilases/genética , DNA Glicosilases/metabolismo , DNA Liase (Sítios Apurínicos ou Apirimidínicos)/genética , Benzimidazóis/farmacologia , Linhagem Celular , DNA/metabolismo , Dano ao DNA/genética , DNA Glicosilases/efeitos dos fármacos , Reparo do DNA/efeitos dos fármacos , Reparo do DNA/genética , DNA Liase (Sítios Apurínicos ou Apirimidínicos)/metabolismo , Guanina/análogos & derivados , Humanos , Cinética , Mutação , Estresse Oxidativo , Piperidinas/farmacologia , Estudos Prospectivos , Espécies Reativas de Oxigênio/metabolismo
12.
Biochemistry ; 60(19): 1485-1497, 2021 05 18.
Artigo em Inglês | MEDLINE | ID: mdl-33929180

RESUMO

Editing of the pre-mRNA of the DNA repair glycosylase NEIL1 results in substitution of a Lys with Arg in the lesion recognition loop of the enzyme. Unedited (UE, Lys242) NEIL1 removes thymine glycol lesions in DNA ∼30 times faster than edited (Ed, Arg242) NEIL1. Herein, we evaluated recognition and excision mediated by UE and Ed NEIL1 of 5-hydroxyuracil (5-OHU), a highly mutagenic lesion formed via oxidation of cytosine. Both NEIL1 isoforms catalyzed low levels of 5-OHU excision in single-stranded DNA, bubble and bulge DNA contexts and in duplex DNA base paired with A. Removal of 5-OHU in base pairs with G, T, and C was found to be faster and proceed to a higher overall extent with UE than with Ed NEIL1. In addition, the presence of mismatches adjacent to 5-OHU magnified the hampered activity of the Ed isoform. However, Ed NEIL1 was found to exhibit higher affinity for 5-OHU:G and 5-OHU:C duplexes than UE NEIL1. These results suggest that NEIL1 plays an important role in detecting and capturing 5-OHU lesions in inappropriate contexts, in a manner that does not lead to excision, to prevent mutations and strand breaks. Indeed, inefficient removal of 5-OHU by NEIL1 from 5-OHU:A base pairs formed during replication would thwart mutagenesis. Notably, nonproductive engagement of 5-OHU by Ed NEIL1 suggests the extent of 5-OHU repair will be reduced under cellular conditions, such as inflammation, that increase the extent of NEIL1 RNA editing. Tipping the balance between the two NEIL1 isoforms may be a significant factor leading to genome instability.


Assuntos
DNA Glicosilases/genética , DNA Glicosilases/metabolismo , Edição de RNA/genética , DNA/genética , Reparo do DNA , DNA de Cadeia Simples , Humanos , Oxirredução , Timina/análogos & derivados , Uracila/análogos & derivados , Uracila/metabolismo
13.
Biochemistry ; 60(11): 873-885, 2021 03 23.
Artigo em Inglês | MEDLINE | ID: mdl-33689312

RESUMO

DNA damage has been implicated in numerous human diseases, particularly cancer, and the aging process. Single-base lesions and mismatches in DNA can be cytotoxic or mutagenic and are recognized by a DNA glycosylase during the process of base excision repair. Altered local dynamics and conformational properties in damaged DNAs have previously been suggested to assist in recognition and specificity. Herein, we use solution nuclear magnetic resonance to quantify changes in BI-BII backbone conformational dynamics due to the presence of single-base lesions in DNA, including uracil, dihydrouracil, 1,N6-ethenoadenine, and T:G mismatches. Stepwise changes to the %BII and ΔG of the BI-BII dynamic equilibrium compared to those of unmodified sequences were observed. Additionally, the equilibrium skews toward endothermicity for the phosphates nearest the lesion/mismatched base pair. Finally, the phosphates with the greatest alterations correlate with those most relevant to the repair of enzyme binding. All of these results suggest local conformational rearrangement of the DNA backbone may play a role in lesion recognition by repair enzymes.


Assuntos
Pareamento Incorreto de Bases , DNA/genética , DNA/metabolismo , Sítios de Ligação , DNA/química , DNA Glicosilases/metabolismo , Reparo do DNA , Humanos , Mutagênese , Conformação de Ácido Nucleico , Ligação Proteica
14.
Nucleic Acids Res ; 49(7): 3948-3966, 2021 04 19.
Artigo em Inglês | MEDLINE | ID: mdl-33784377

RESUMO

Uracil occurs at replication forks via misincorporation of deoxyuridine monophosphate (dUMP) or via deamination of existing cytosines, which occurs 2-3 orders of magnitude faster in ssDNA than in dsDNA and is 100% miscoding. Tethering of UNG2 to proliferating cell nuclear antigen (PCNA) allows rapid post-replicative removal of misincorporated uracil, but potential 'pre-replicative' removal of deaminated cytosines in ssDNA has been questioned since this could mediate mutagenic translesion synthesis and induction of double-strand breaks. Here, we demonstrate that uracil-DNA glycosylase (UNG), but not SMUG1 efficiently excises uracil from replication protein A (RPA)-coated ssDNA and that this depends on functional interaction between the flexible winged-helix (WH) domain of RPA2 and the N-terminal RPA-binding helix in UNG. This functional interaction is promoted by mono-ubiquitination and diminished by cell-cycle regulated phosphorylations on UNG. Six other human proteins bind the RPA2-WH domain, all of which are involved in DNA repair and replication fork remodelling. Based on this and the recent discovery of the AP site crosslinking protein HMCES, we propose an integrated model in which templated repair of uracil and potentially other mutagenic base lesions in ssDNA at the replication fork, is orchestrated by RPA. The UNG:RPA2-WH interaction may also play a role in adaptive immunity by promoting efficient excision of AID-induced uracils in transcribed immunoglobulin loci.


Assuntos
DNA Glicosilases/metabolismo , Replicação do DNA , DNA de Cadeia Simples/metabolismo , Proteína de Replicação A/metabolismo , Uracila/metabolismo , Sítios de Ligação , Humanos , Ligação Proteica , Proteínas Recombinantes/metabolismo
15.
Prog Biophys Mol Biol ; 164: 72-80, 2021 09.
Artigo em Inglês | MEDLINE | ID: mdl-33753087

RESUMO

Cell survival largely depends on the faithful maintenance of genetic material since genomic DNA is constantly exposed to genotoxicants from both endogenous and exogenous sources. The evolutionarily conserved base excision repair (BER) pathway is critical for maintaining genome integrity by eliminating highly abundant and potentially mutagenic oxidized DNA base lesions. BER is a multistep process, which is initiated with recognition and excision of the DNA base lesion by a DNA glycosylase, followed by DNA end processing, gap filling and finally sealing of the nick. Besides genome maintenance by global BER, DNA glycosylases have been found to play additional roles, including preferential repair of oxidized lesions from transcribed genes, modulation of the immune response, participation in active DNA demethylation and maintenance of the mitochondrial genome. Central to these functions is the DNA glycosylase NEIL2. Its loss results in increased accumulation of oxidized base lesions in the transcribed genome, triggers an immune response and causes early neurodevelopmental defects, thus emphasizing the multitasking capabilities of this repair protein. Here we review the specialized functions of NEIL2 and discuss the consequences of its absence both in vitro and in vivo.


Assuntos
DNA Glicosilases , DNA , Dano ao DNA , DNA Glicosilases/genética , DNA Glicosilases/metabolismo , Reparo do DNA , DNA Liase (Sítios Apurínicos ou Apirimidínicos)/genética , DNA Liase (Sítios Apurínicos ou Apirimidínicos)/metabolismo
16.
DNA Repair (Amst) ; 99: 103051, 2021 03.
Artigo em Inglês | MEDLINE | ID: mdl-33540225

RESUMO

The integrity of the genetic information is continuously challenged by numerous genotoxic insults, most frequently in the form of oxidation, alkylation or deamination of the bases that result in DNA damage. These damages compromise the fidelity of the replication, and interfere with the progression and function of the transcription machineries. The DNA damage response (DDR) comprises a series of strategies to deal with DNA damage, including transient transcriptional inhibition, activation of DNA repair pathways and chromatin remodeling. Coordinated control of transcription and DNA repair is required to safeguardi cellular functions and identities. Here, we address the cellular responses to endogenous DNA damage, with a particular focus on the role of DNA glycosylases and the Base Excision Repair (BER) pathway, in conjunction with the DDR factors, in responding to DNA damage during the transcription process. We will also discuss functions of newly identified epigenetic and regulatory marks, such as 5-hydroxymethylcytosine and its oxidative products and 8-oxoguanine, that were previously considered only as DNA damages. In light of these resultsthe classical perception of DNA damage as detrimental for cellular processes are changing. and a picture emerges whereDNA glycosylases act as dynamic regulators of transcription, placing them at the intersection of DNA repair and gene expression modulation.


Assuntos
Dano ao DNA , DNA Glicosilases/metabolismo , Reparo do DNA , Epigênese Genética , 5-Metilcitosina/análogos & derivados , Animais , DNA/metabolismo , Regulação da Expressão Gênica , Guanina/análogos & derivados , Humanos
17.
Int J Mol Sci ; 22(3)2021 Jan 29.
Artigo em Inglês | MEDLINE | ID: mdl-33573045

RESUMO

X-ray analysis cannot provide quantitative estimates of the relative contribution of non-specific, specific, strong, and weak contacts of extended DNA molecules to their total affinity for enzymes and proteins. The interaction of different enzymes and proteins with long DNA and RNA at the quantitative molecular level can be successfully analyzed using the method of the stepwise increase in ligand complexity (SILC). The present review summarizes the data on stepwise increase in ligand complexity (SILC) analysis of nucleic acid recognition by various enzymes-replication, restriction, integration, topoisomerization, six different repair enzymes (uracil DNA glycosylase, Fpg protein from Escherichia coli, human 8-oxoguanine-DNA glycosylase, human apurinic/apyrimidinic endonuclease, RecA protein, and DNA-ligase), and five DNA-recognizing proteins (RNA helicase, human lactoferrin, alfa-lactalbumin, human blood albumin, and IgGs against DNA). The relative contributions of structural elements of DNA fragments "covered" by globules of enzymes and proteins to the total affinity of DNA have been evaluated. Thermodynamic and catalytic factors providing discrimination of unspecific and specific DNAs by these enzymes on the stages of primary complex formation following changes in enzymes and DNAs or RNAs conformations and direct processing of the catalysis of the reactions were found. General regularities of recognition of nucleic acid by DNA-dependent enzymes, proteins, and antibodies were established.


Assuntos
Anticorpos/metabolismo , DNA/metabolismo , Proteínas/metabolismo , Animais , DNA Glicosilases/metabolismo , DNA Helicases/metabolismo , DNA Ligases/metabolismo , DNA Topoisomerases Tipo I/metabolismo , DNA Liase (Sítios Apurínicos ou Apirimidínicos)/metabolismo , DNA Polimerase Dirigida por DNA/metabolismo , Desoxirribonuclease EcoRI/metabolismo , Humanos , Lactalbumina/metabolismo , Lactoferrina/metabolismo , Recombinases Rec A/metabolismo , Albumina Sérica Humana/metabolismo
18.
Chem Res Toxicol ; 34(3): 901-911, 2021 03 15.
Artigo em Inglês | MEDLINE | ID: mdl-33595290

RESUMO

Dietary exposure to aflatoxins is a significant risk factor in the development of hepatocellular carcinomas. Following bioactivation by microsomal P450s, the reaction of aflatoxin B1 (AFB1) with guanine (Gua) in DNA leads to the formation of stable, imidazole ring-opened 8,9-dihydro-8-(2,6-diamino-4-oxo-3,4-dihydropyrimid-5-yl-formamido)-9-hydroxyaflatoxin B1 (AFB1-FapyGua) adducts. In contrast to most base modifications that result in destabilization of the DNA duplex, the AFB1-FapyGua adduct increases the thermal stability of DNA via 5'-interface intercalation and base-stacking interactions. Although it was anticipated that this stabilization might make these lesions difficult to repair relative to helix distorting modifications, prior studies have shown that both the nucleotide and base excision repair pathways participate in the removal of the AFB1-FapyGua adduct. Specifically for base excision repair, we previously showed that the DNA glycosylase NEIL1 excises AFB1-FapyGua and catalyzes strand scission in both synthetic oligodeoxynucleotides and liver DNA of exposed mice. Since it is anticipated that error-prone replication bypass of unrepaired AFB1-FapyGua adducts contributes to cellular transformation and carcinogenesis, the structural and thermodynamic parameters that modulate the efficiencies of these repair pathways are of considerable interest. We hypothesized that the DNA sequence context in which the AFB1-FapyGua adduct is formed might modulate duplex stability and consequently alter the efficiencies of NEIL1-initiated repair. To address this hypothesis, site-specific AFB1-FapyGua adducts were synthesized in three sequence contexts, with the 5' neighbor nucleotide being varied. DNA structural stability analyses were conducted using UV absorbance- and NMR-based melting experiments. These data revealed differentials in thermal stabilities associated with the 5'-neighbor base pair. Single turnover kinetic analyses using the NEIL1 glycosylase demonstrated corresponding sequence-dependent differences in the repair of this adduct, such that there was an inverse correlation between the stabilization of the duplex and the efficiency of NEIL1-mediated catalysis.


Assuntos
Aflatoxina B1/metabolismo , Adutos de DNA/metabolismo , DNA Glicosilases/metabolismo , DNA/metabolismo , Guanina/metabolismo , Pirimidinas/metabolismo , Aflatoxina B1/química , Sequência de Bases , Biocatálise , DNA/química , Adutos de DNA/química , DNA Glicosilases/química , Guanina/química , Humanos , Estrutura Molecular , Pirimidinas/química
19.
Sci Rep ; 11(1): 3490, 2021 02 10.
Artigo em Inglês | MEDLINE | ID: mdl-33568707

RESUMO

The most common oxidative DNA lesion is 8-oxoguanine which is mainly recognized and excised by the 8-oxoG DNA glycosylase 1 (OGG1), initiating the base excision repair (BER) pathway. Telomeres are particularly sensitive to oxidative stress (OS) which disrupts telomere homeostasis triggering genome instability. In the present study, we have investigated the effects of inactivating BER in OS conditions, by using a specific inhibitor of OGG1 (TH5487). We have found that in OS conditions, TH5487 blocks BER initiation at telomeres causing an accumulation of oxidized bases, that is correlated with telomere losses, micronuclei formation and mild proliferation defects. Moreover, the antimetabolite methotrexate synergizes with TH5487 through induction of intracellular reactive oxygen species (ROS) formation, which potentiates TH5487-mediated telomere and genome instability. Our findings demonstrate that OGG1 is required to protect telomeres from OS and present OGG1 inhibitors as a tool to induce oxidative DNA damage at telomeres, with the potential for developing new combination therapies for cancer treatment.


Assuntos
Antimetabólitos Antineoplásicos/farmacologia , Benzimidazóis/farmacologia , DNA Glicosilases/antagonistas & inibidores , Reparo do DNA/efeitos dos fármacos , Metotrexato/farmacologia , Estresse Oxidativo , Piperidinas/farmacologia , Telômero/metabolismo , Ciclo Celular , Linhagem Celular Tumoral , DNA Glicosilases/metabolismo , Sinergismo Farmacológico , Inibidores Enzimáticos/farmacologia , Instabilidade Genômica , Humanos , Oxirredução , Espécies Reativas de Oxigênio/metabolismo
20.
Sci Rep ; 11(1): 859, 2021 01 13.
Artigo em Inglês | MEDLINE | ID: mdl-33441677

RESUMO

While most restriction enzymes catalyze the hydrolysis of phosphodiester bonds at specific nucleotide sequences in DNA, restriction enzymes of the HALFPIPE superfamily cleave N-glycosidic bonds, similar to DNA glycosylases. Apurinic/apyrimidinic (AP) sites generated by HALFPIPE superfamily proteins are cleaved by their inherent AP lyase activities, other AP endonuclease activities or heat-promoted ß-elimination. Although the HALFPIPE superfamily protein R.PabI, obtained from a hyperthermophilic archaea, Pyrococcus abyssi, shows weak AP lyase activity, HALFPIPE superfamily proteins in mesophiles, such as R.CcoLI from Campylobacter coli and R. HpyAXII from Helicobacter pylori, show significant AP lyase activities. To identify the structural basis for the AP lyase activity of R.CcoLI, we determined the structure of R.CcoLI by X-ray crystallography. The structure of R.CcoLI, obtained at 2.35-Å resolution, shows that a conserved lysine residue (Lys71), which is stabilized by a characteristic ß-sheet structure of R.CcoLI, protrudes into the active site. The results of mutational assays indicate that Lys71 is important for the AP lyase activity of R.CcoLI. Our results help to elucidate the mechanism by which HALFPIPE superfamily proteins from mesophiles efficiently introduce double-strand breaks to specific sites on double-stranded DNA.


Assuntos
Campylobacter coli/enzimologia , DNA Glicosilases/genética , DNA Glicosilases/ultraestrutura , Sequência de Bases/genética , Campylobacter coli/genética , Campylobacter coli/metabolismo , Domínio Catalítico/genética , Cristalografia por Raios X/métodos , DNA/química , DNA/genética , Clivagem do DNA , Dano ao DNA/genética , DNA Glicosilases/metabolismo , Reparo do DNA/genética , Enzimas de Restrição do DNA/metabolismo , Enzimas de Restrição do DNA/ultraestrutura , DNA Liase (Sítios Apurínicos ou Apirimidínicos)/metabolismo , DNA Liase (Sítios Apurínicos ou Apirimidínicos)/ultraestrutura
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