RESUMO
Reactive oxygen species induce oxidative damage in DNA precursors, i.e. dNTPs, leading to point mutations upon incorporation. Escherichia coli mutT strains, deficient in the activity hydrolysing 8-oxo-7,8-dihydro-2'-deoxyguanosine 5'-triphosphate (8-oxo-dGTP), display more than a 100-fold higher spontaneous mutation frequency over the wild-type strain. 8-oxo-dGTP induces A to C transversions when misincorporated opposite template A. Here, we report that DNA pol III incorporates 8-oxo-dGTP ≈ 20 times more efficiently opposite template A compared with template C. Single, double or triple deletions of pol I, pol II, pol IV or pol V had modest effects on the mutT mutator phenotype. Only the deletion of all four polymerases led to a 70% reduction of the mutator phenotype. While pol III may account for nearly all 8-oxo-dGTP incorporation opposite template A, it only extends ≈ 30% of them, the remaining 70% being extended by the combined action of pol I, pol II, pol IV or pol V. The unique property of pol III, a C-family DNA polymerase present only in eubacteria, to preferentially incorporate 8-oxo-dGTP opposite template A during replication might explain the high spontaneous mutation frequency in E. coli mutT compared with the mammalian counterparts lacking the 8-oxo-dGTP hydrolysing activities.
Assuntos
DNA Polimerase III/metabolismo , Escherichia coli/enzimologia , Escherichia coli/genética , Taxa de Mutação , Mutação , Pirofosfatases/deficiência , DNA Bacteriano/metabolismo , Nucleotídeos de Desoxiguanina/metabolismo , Proteínas de Escherichia coliRESUMO
Oxidized DNA precursors can cause mutagenesis and carcinogenesis when they are incorporated into the genome. Some human Y-family DNA polymerases (Pols) can effectively incorporate 8-oxo-dGTP, an oxidized form of dGTP, into a position opposite a template dA. This inappropriate G:A pairing may lead to transversions of A to C. To gain insight into the mechanisms underlying erroneous nucleotide incorporation, we changed amino acids in human Poleta and Polkappa proteins that might modulate their specificity for incorporating 8-oxo-dGTP into DNA. We found that Arg61 in Poleta was crucial for erroneous nucleotide incorporation. When Arg61 was substituted with lysine (R61K), the ratio of pairing of dA to 8-oxo-dGTP compared to pairing of dC was reduced from 660:1 (wild-type Poleta) to 7 : 1 (R61K). Similarly, Tyr112 in Polkappa was crucial for erroneous nucleotide incorporation. When Tyr112 was substituted with alanine (Y112A), the ratio of pairing was reduced from 11: 1 (wild-type Polkappa) to almost 1: 1 (Y112A). Interestingly, substitution at the corresponding position in Poleta, i.e. Phe18 to alanine, did not alter the specificity. These results suggested that amino acids at distinct positions in the active sites of Poleta and Polkappa might enhance 8-oxo-dGTP to favor the syn conformation, and thus direct its misincorporation into DNA.
Assuntos
DNA Polimerase Dirigida por DNA/química , Nucleotídeos de Desoxiguanina/química , Substituição de Aminoácidos , Arginina/genética , Pareamento de Bases , Domínio Catalítico , DNA/biossíntese , DNA/química , DNA Polimerase Dirigida por DNA/genética , DNA Polimerase Dirigida por DNA/metabolismo , Desoxiadenosinas/química , Nucleotídeos de Desoxiguanina/metabolismo , Humanos , Cinética , Modelos Moleculares , OxirreduçãoRESUMO
Human cells possess multiple specialized DNA polymerases (Pols) that bypass a variety of DNA lesions which otherwise would block chromosome replication. Human polymerase kappa (Pol κ) bypasses benzo[a]pyrene diolepoxide-N(2)-deoxyguanine (BPDE-N(2)-dG) DNA adducts in an almost error-free manner. To better understand the relationship between the structural features in the active site and lesion bypass by Pol κ, we mutated codons corresponding to amino acids appearing close to the adducts in the active site, and compared bypass efficiencies. Remarkably, the substitution of alanine for phenylalanine 171 (F171), an amino acid conserved between Pol κ and its bacterial counterpart Escherichia coli DinB, enhanced the efficiencies of dCMP incorporation opposite (-)- and (+)-trans-anti-BPDE-N(2)-dG 18-fold. This substitution affected neither the fidelity of TLS nor the efficiency of dCMP incorporation opposite normal guanine. This amino acid change also enhanced the binding affinity of Pol κ to template/primer DNA containing (-)-trans-anti-BPDE-N(2)-dG. These results suggest that F171 functions as a molecular brake for TLS across BPDE-N(2)-dG by Pol κ and that the F171A derivative of Pol κ bypasses these DNA lesions more actively than does the wild-type enzyme.
Assuntos
Benzo(a)pireno/metabolismo , Adutos de DNA/metabolismo , DNA Polimerase Dirigida por DNA/química , DNA Polimerase Dirigida por DNA/metabolismo , 7,8-Di-Hidro-7,8-Di-Hidroxibenzo(a)pireno 9,10-óxido/análogos & derivados , 7,8-Di-Hidro-7,8-Di-Hidroxibenzo(a)pireno 9,10-óxido/química , 7,8-Di-Hidro-7,8-Di-Hidroxibenzo(a)pireno 9,10-óxido/metabolismo , Substituição de Aminoácidos , Sequência de Bases , Benzo(a)pireno/química , Domínio Catalítico/genética , Adutos de DNA/química , Dano ao DNA , Primers do DNA/genética , Reparo do DNA , DNA Polimerase Dirigida por DNA/genética , Desoxiguanosina/análogos & derivados , Desoxiguanosina/química , Desoxiguanosina/metabolismo , Proteínas de Escherichia coli/química , Proteínas de Escherichia coli/genética , Proteínas de Escherichia coli/metabolismo , Humanos , Técnicas In Vitro , Cinética , Modelos Moleculares , Mutagênese Sítio-Dirigida , Fenilalanina/química , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Especificidade por SubstratoRESUMO
DNA-protein cross-links (DPCs) are unique among DNA lesions in their unusually bulky nature. The steric hindrance imposed by cross-linked proteins (CLPs) will hamper DNA transactions, such as replication and transcription, posing an enormous threat to cells. In bacteria, DPCs with small CLPs are eliminated by nucleotide excision repair (NER), whereas oversized DPCs are processed exclusively by RecBCD-dependent homologous recombination (HR). Here we have assessed the roles of NER and HR for DPCs in mammalian cells. We show that the upper size limit of CLPs amenable to mammalian NER is relatively small (8-10 kDa) so that NER cannot participate in the repair of chromosomal DPCs in mammalian cells. Moreover, CLPs are not polyubiquitinated and hence are not subjected to proteasomal degradation prior to NER. In contrast, HR constitutes the major pathway in tolerance of DPCs as judged from cell survival and RAD51 and gamma-H2AX nuclear foci formation. Induction of DPCs results in the accumulation of DNA double strand breaks in HR-deficient but not HR-proficient cells, suggesting that fork breakage at the DPC site initiates HR and reactivates the stalled fork. DPCs activate both ATR and ATM damage response pathways, but there is a time lag between two responses. These results highlight the differential involvement of NER in the repair of DPCs in bacterial and mammalian cells and demonstrate the versatile and conserved role of HR in tolerance of DPCs among species.
Assuntos
Reagentes de Ligações Cruzadas/farmacologia , Reparo do DNA , DNA/metabolismo , Desoxirribonucleotídeos/genética , Proteínas/metabolismo , Recombinação Genética , Animais , Ataxia Telangiectasia/genética , Ataxia Telangiectasia/metabolismo , Azacitidina/análogos & derivados , Azacitidina/farmacologia , Proteína BRCA2/metabolismo , Sequência de Bases , Proteínas de Ciclo Celular/metabolismo , Linhagem Celular , Cromossomos/metabolismo , Cricetinae , DNA/química , DNA/genética , Quebras de DNA de Cadeia Dupla/efeitos dos fármacos , Decitabina , Escherichia coli/citologia , Escherichia coli/genética , Escherichia coli/metabolismo , Proteína do Grupo de Complementação D2 da Anemia de Fanconi/metabolismo , Formaldeído/farmacologia , Histonas/metabolismo , Humanos , Peso Molecular , Mutação , Complexo de Endopeptidases do Proteassoma/metabolismo , Proteínas/química , Rad51 Recombinase/metabolismoRESUMO
Genetic information must be duplicated with precision and accurately passed on to daughter cells and later generations. In order to achieve this goal, DNA polymerases (Pols) have to faithfully execute DNA synthesis during chromosome replication and repair. However, the conditions under which Pols synthesize DNA are not always optimal; the template DNA can be damaged by various endogenous and exogenous genotoxic agents including reactive oxygen species (ROS), and ROS oxidize dNTPs in the nucleotide pool from which Pols elongate DNA strands. Both damaged DNA and oxidized dNTPs interfere with faithful DNA synthesis by Pols, inducing various cellular abnormalities, such as mutations, cancer, neurological diseases, and cellular senescence. In this review, we focus on the process by which Pols incorporate oxidized dNTPs into DNA and compare the properties of Pols: efficiency, i.e., k(cat)/K(m), k(pol)/K(d) or V(max)/K(m), and template base preference for the incorporation of 8-oxo-dGTP, an oxidized form of dGTP. In general, Pols involved in chromosome replication, the A- and B-family Pols, are resistant to the incorporation of 8-oxo-dGTP, whereas Pols involved in repair and/or translesion synthesis, the X- and Y-family Pols, incorporate nucleotides in a relatively efficient manner and tend to incorporate it opposite template dA rather than template dC, though there are several exceptions. We discuss the molecular mechanisms by which Pols exhibit different template base preferences for the incorporation of 8-oxo-dGTP and how Pols are involved in the induction of mutations via the incorporation of oxidized nucleotides under oxidative stress.
Assuntos
Dano ao DNA , DNA Polimerase Dirigida por DNA/metabolismo , Nucleotídeos de Desoxiguanina/metabolismo , Espécies Reativas de Oxigênio , Animais , Reparo do DNA , Enzimas Reparadoras do DNA/metabolismo , Nucleotídeos de Desoxiguanina/química , Humanos , Monoéster Fosfórico Hidrolases/metabolismo , Moldes GenéticosRESUMO
Human DNA is continuously damaged by exogenous and endogenous genotoxic insults. To counteract DNA damage and ensure the completion of DNA replication, cells possess specialized DNA polymerases (Pols) that bypass a variety of DNA lesions. Human DNA polymerase kappa (hPolkappa) is a member of the Y-family of DNA Pols and a direct counterpart of DinB in Escherichia coli. hPolkappa is characterized by its ability to bypass several DNA adducts [e.g., benzo[a]pyrene diolepoxide-N(2)-deoxyguanine (BPDE-N(2)-dG) and thymine glycol] and efficiently extend primers with mismatches at the termini. hPolkappa is structurally distinct from E. coli DinB in that it possesses an approximately 100-amino acid extension at the N-terminus. Here, we report that tyrosine 112 (Y112), the steric gate amino acid of hPolkappa, which distinguishes dNTPs from rNTPs by sensing the 2'-hydroxy group of incoming nucleotides, plays a crucial role in extension reactions with mismatched primer termini. When Y112 was replaced with alanine, the amino acid change severely reduced the catalytic constant, i.e., k(cat), of the extending mismatched primers and lowered the efficiency, i.e., k(cat)/K(m), of this process by approximately 400-fold compared with that of the wild-type enzyme. In contrast, the amino acid replacement did not reduce the insertion efficiency of dCMP opposite BPDE-N(2)-dG in template DNA, nor did it affect the ability of hPolkappa to bind strongly to template-primer DNA with BPDE-N(2)-dG/dCMP. We conclude that the steric gate of hPolkappa is a major fidelity factor that regulates extension reactions from mismatched primer termini.
Assuntos
Primers do DNA/química , DNA Polimerase Dirigida por DNA/química , Tirosina/química , Aminoácidos/química , Pareamento Incorreto de Bases , Catálise , Adutos de DNA , Replicação do DNA , Escherichia coli/metabolismo , Proteínas de Escherichia coli/metabolismo , Vetores Genéticos , Humanos , Cinética , Modelos Moleculares , MutaçãoRESUMO
SUMMARY: Upon blockage of chromosomal replication by DNA lesions, Y-family polymerases interact with monoubiquitylated proliferating cell nuclear antigen (PCNA) to catalyse translesion synthesis (TLS) and restore replication fork progression. Here, we assessed the roles of Arabidopsis thaliana POLH, which encodes a homologue of Y-family polymerase eta (Poleta), PCNA1 and PCNA2 in TLS-mediated UV resistance. A T-DNA insertion in POLH sensitized the growth of roots and whole plants to UV radiation, indicating that AtPoleta contributes to UV resistance. POLH alone did not complement the UV sensitivity conferred by deletion of yeast RAD30, which encodes Poleta, although AtPoleta exhibited cyclobutane dimer bypass activity in vitro, and interacted with yeast PCNA, as well as with Arabidopsis PCNA1 and PCNA2. Co-expression of POLH and PCNA2, but not PCNA1, restored normal UV resistance and mutation kinetics in the rad30 mutant. A single residue difference at site 201, which lies adjacent to the residue (lysine 164) ubiquitylated in PCNA, appeared responsible for the inability of PCNA1 to function with AtPoleta in UV-treated yeast. PCNA-interacting protein boxes and an ubiquitin-binding motif in AtPoleta were found to be required for the restoration of UV resistance in the rad30 mutant by POLH and PCNA2. These observations indicate that AtPoleta can catalyse TLS past UV-induced DNA damage, and links the biological activity of AtPoleta in UV-irradiated cells to PCNA2 and PCNA- and ubiquitin-binding motifs in AtPoleta.
Assuntos
Proteínas de Arabidopsis/metabolismo , Arabidopsis/genética , Reparo do DNA , DNA Polimerase Dirigida por DNA/metabolismo , Antígeno Nuclear de Célula em Proliferação/metabolismo , Sequência de Aminoácidos , Arabidopsis/metabolismo , Arabidopsis/efeitos da radiação , Proteínas de Arabidopsis/genética , Clonagem Molecular , Dano ao DNA , Replicação do DNA , DNA Bacteriano/genética , DNA Complementar/genética , DNA Polimerase Dirigida por DNA/genética , Teste de Complementação Genética , Dados de Sequência Molecular , Mutagênese Insercional , Mutação , Raízes de Plantas/genética , Raízes de Plantas/metabolismo , Raízes de Plantas/efeitos da radiação , RNA de Plantas/genética , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Alinhamento de Sequência , Técnicas do Sistema de Duplo-Híbrido , Raios UltravioletaRESUMO
Chromosomal translocation is a key process in the oncogenic transformation of somatic cells. Previously, artificial induction of chromosomal translocation was performed using homologous recombination-mediated loxP labeling of target regions followed by Cre-mediated recombination. Recent progress in genome editing techniques has facilitated the easier induction of artificial translocation by cutting two targeted genome sequences from different chromosomes. The present study established a system to induce t(11;14)(q13;q32), which is observed primarily in multiple myeloma (MM) and involves the repositioning of the cyclin D1 (CCND1) gene downstream of the immunoglobulin heavy chain (IgH) constant region enhancers by translocation. The placing of tandem gRNAs designed to cut both the IgH Eµ and CCND1 15-kb upstream regions in lentiCRISPRv2 enabled the induction of chromosomal translocation in 293T cells, with confirmation by translocation-specific PCR and fluorescence in situ hybridization probing with IgH and CCND1. At the translocation junctions, small deletions and the addition of DNA sequences (indels) were observed in several clones. Cloned cells with t(11;14) exhibited slower growth and lower CCND1 mRNA expression compared to the parent cells, presenting the opposite phenomena induced by t(11;14) in MM cells, indicating that the silent IgH gene juxtaposed to CCND1 may negatively affect CCND1 gene expression and cell proliferation in the non-B lymphocyte lineage. Therefore, the present study achieved the induction of silent promoter/enhancer translocation in t(11;14)(q13;q32) as a preparatory experiment to study the role of IgH constant region enhancer-driven CCND1 overexpression in oncogenic transformation processes in B lymphocytes.
RESUMO
Endonuclease III (Endo III) is a base excision repair enzyme that recognizes oxidized pyrimidine bases including thymine glycol. This enzyme is a glycosylase/lyase and forms a Schiff base-type intermediate with the substrate after the damaged base is removed. To investigate the mechanism of its substrate recognition by X-ray crystallography, we have synthesized oligonucleotides containing 2'-fluorothymidine glycol, expecting that the electron-withdrawing fluorine atom at the 2' position would stabilize the covalent intermediate, as observed for T4 endonuclease V (Endo V) in our previous study. Oxidation of 5'- and 3'-protected 2'-fluorothymidine with OsO4 produced two isomers of thymine glycol. Their configurations were determined by NMR spectroscopy after protection of the hydroxyl functions. The ratio of (5R,6S) and (5S,6R) isomers was 3:1, whereas this ratio was 6:1 in the case of the unmodified sugar. Both of the thymidine glycol isomers were converted to the corresponding phosphoramidite building blocks and were incorporated into oligonucleotides. When the duplexes containing 2'-fluorinated 5R- or 5S-thymidine glycol were treated with Escherichia coli endo III, no stabilized covalent intermediate was observed regardless of the stereochemistry at C5. The 5S isomer was found to form an enzyme-DNA complex, but the incision was inhibited probably by the fluorine-induced stabilization of the glycosidic bond.
Assuntos
Desoxirribonuclease (Dímero de Pirimidina)/metabolismo , Inibidores Enzimáticos/química , Proteínas de Escherichia coli/metabolismo , Oligonucleotídeos/síntese química , Timidina/análogos & derivados , Inibidores Enzimáticos/síntese química , Isomerismo , Oligonucleotídeos/química , Oligonucleotídeos/metabolismo , Compostos Organofosforados/síntese química , Compostos Organofosforados/química , Especificidade por Substrato , Timidina/química , Proteínas Virais/metabolismoRESUMO
Nitric oxide (NO) induces deamination of guanine, yielding xanthine and oxanine (Oxa). Furthermore, Oxa reacts with polyamines and DNA binding proteins to form cross-link adducts. Thus, it is of interest how these lesions are processed by DNA repair enzymes in view of the genotoxic mechanism of NO. In the present study, we have examined the repair capacity for Oxa and Oxa-spermine cross-link adducts (Oxa-Sp) of enzymes involved in base excision repair (BER) and nucleotide excision repair (NER) to delineate the repair mechanism of nitrosative damage to guanine. Oligonucleotide substrates containing Oxa and Oxa-Sp were incubated with purified BER and NER enzymes or cell-free extracts (CFEs), and the damage-excising or DNA-incising activity was compared with that for control (physiological) substrates. The Oxa-excising activities of Escherichia coli and human DNA glycosylases and HeLa CFEs were 0.2-9% relative to control substrates, implying poor processing of Oxa by BER. In contrast, DNA containing Oxa-Sp was incised efficiently by UvrABC nuclease and SOS-induced E.coli CFEs, suggesting a role of NER in ameliorating genotoxic effects associated with nitrosative stress. Analyses of the activity of CFEs from NER-proficient and NER-deficient human cells on Oxa-Sp DNA confirmed further the involvement of NER in the repair of nitrosative DNA damage.
Assuntos
Enzimas Reparadoras do DNA/metabolismo , Reparo do DNA , Óxido Nítrico/toxicidade , Nucleosídeos de Purina/metabolismo , Espermina/metabolismo , Sequência de Bases , Extratos Celulares , Adutos de DNA/metabolismo , Dano ao DNA , DNA Glicosilases/metabolismo , Endodesoxirribonucleases/metabolismo , Escherichia coli/enzimologia , Proteínas de Escherichia coli/metabolismo , Células HeLa , Humanos , Dados de Sequência Molecular , Oligonucleotídeos/química , Oligonucleotídeos/metabolismoRESUMO
B cell derived induced pluripotent stem cells (BiPSCs) were recently established from peripheral blood B cells by the simultaneous transfection of Yamanaka factors (Oct3/4, Sox2, Klf4, c-Myc) and C/EBPα using a Sendai virus vector. Here, using a different method, we established BiPSCs with immunoglobulin heavy chain (IgH) gene rearrangement from normal B cells purified from lymph nodes. The critical points of our method are pre-stimulation of B cells with IL-21 and CD40-ligand (CD40L), followed by consecutive transfection of highly concentrated Yamanaka factors using a retroviral vector. Following each transfection the cells were centrifuged onto a retronectin coated plate and the activated by IL-4, IL-2, and CD40L. Furthermore, we established BiPSCs (BiPSC-A) in which activation-induced cytidine deaminase (AID) could be induced using the doxycycline-controlled. Both the parental BiPSC and BiPSC-A showed the capability of differentiating into hematopoietic progenitor cells (HPCs) based on confirmation of CD34 expression and colony-formation from CD34-positive cells. The findings that BiPSC-A can differentiate into HPCs suggest that there is a possibility that induction of AID expression would result in chromosomal translocations in the process of differentiation from BiPSCs, and therefore that these BiPSCs could be useful in elucidating the tumor origin of abnormal B cells in myelomagenesis.
Assuntos
Linfócitos B/citologia , Diferenciação Celular , Citidina Desaminase/biossíntese , Células-Tronco Hematopoéticas/citologia , Células-Tronco Pluripotentes Induzidas/citologia , Animais , Antígenos CD19/metabolismo , Linfócitos B/efeitos dos fármacos , Linfócitos B/metabolismo , Diferenciação Celular/efeitos dos fármacos , Ensaio de Unidades Formadoras de Colônias , Doxiciclina/farmacologia , Indução Enzimática/efeitos dos fármacos , Hematopoese/efeitos dos fármacos , Células-Tronco Hematopoéticas/efeitos dos fármacos , Células-Tronco Hematopoéticas/metabolismo , Humanos , Células-Tronco Pluripotentes Induzidas/efeitos dos fármacos , Células-Tronco Pluripotentes Induzidas/metabolismo , Fator 4 Semelhante a Kruppel , Linfonodos/citologia , Camundongos SCID , Modelos BiológicosRESUMO
We recently reported an increase in dicentric chromosome (DIC) formation after a single computed tomography (CT) scan (5.78-60.27 mSv: mean 24.24 mSv) and we recommended analysis of 2000 metaphase cells stained with Giemsa and centromere-FISH for dicentric chromosome assay (DCA) in cases of low-dose radiation exposure. In the present study, we analyzed the frequency of chromosome translocations using stored Carnoy's-fixed lymphocyte specimens from the previous study; these specimens were from 12 patients who were subject to chromosome painting of Chromosomes 1, 2 and 4. Chromosomes 1, 2 and 4 were analyzed in â¼5000 cells, which is equivalent to the whole-genome analysis of almost 2000 cells. The frequency of chromosome translocation was higher than the number of DICs formed, both before and after CT scanning. The frequency of chromosome translocations tended to be higher, but not significantly higher, in patients with a treatment history compared with patients without such a history. However, in contrast to the results for DIC formation, the frequency of translocations detected before and after the CT scan did not differ significantly. Therefore, analysis of chromosome translocation may not be a suitable assay for detecting chromosome aberrations in cases of low-dose radiation exposure from a CT scan. A significant increase in the frequency of chromosome translocations was not likely to be detected due to the high baseline before the CT scan; the high and variable frequency of translocations was probably due to multiple confounding factors in adults.
Assuntos
Cromossomos Humanos/genética , Tomografia Computadorizada por Raios X , Translocação Genética , Adulto , Idoso , Idoso de 80 Anos ou mais , Feminino , Humanos , Masculino , Pessoa de Meia-IdadeRESUMO
Ionizing radiation (IR) induces cellular stress responses, such as signal transduction, gene expression, protein modification, and metabolite change that affect cellular behavior. We analyzed X-irradiated human Epstein-Barr virus-transformed B lymphoblastoid cells and normal fibroblasts to search for metabolites that would be suitable IR-responsive markers by Liquid Chromotography-Mass spectrometry (LC-MS). Mass spectra, as analyzed with principal component analysis, showed that the proportion of peaks with IR-induced change was relatively small compared with the influence of culture time. Dozens of peaks that had either been upregulated or downregulated by IR were extracted as candidate IR markers. The IR-changed peaks were identified by comparing mock-treated groups to 100 mGy-irradiated groups that had recovered after 10 h, and the results indicated that the metabolites involved in nucleoside synthesis increased and that some acylcarnitine levels decreased in B lymphoblastoids. Some peaks changed by as much as 20 mGy, indicating the presence of an IR-sensitive signal transduction/metabolism control mechanism in these cells. On the other hand, we could not find common IR-changed peaks in fibroblasts of different origin. These data suggest that cell phenotype-specific pathways exist, even in low-dose responses, and could determine cell behavior.
Assuntos
Fibroblastos/metabolismo , Fibroblastos/efeitos da radiação , Ácidos Nucleicos/metabolismo , Células Precursoras de Linfócitos B/metabolismo , Células Precursoras de Linfócitos B/efeitos da radiação , Transdução de Sinais/fisiologia , Relação Dose-Resposta à Radiação , Humanos , Doses de Radiação , Transdução de Sinais/efeitos da radiação , Raios XRESUMO
Excess risk of leukemia and brain tumors after CT scans in children has been reported. We performed dicentric chromosome assay (DCAs) before and after CT scan to assess effects of low-dose ionizing radiation on chromosomes. Peripheral blood (PB) lymphocytes were collected from 10 patients before and after a CT scan. DCA was performed by analyzing either 1,000 or 2,000 metaphases using both Giemsa staining and centromere-fluorescence in situ hybridization (Centromere-FISH). The increment of DIC formation was compared with effective radiation dose calculated using the computational dosimetry system, WAZA-ARI and dose length product (DLP) in a CT scan. Dicentric chromosome (DIC) formation increased significantly after a single CT scan, and increased DIC formation was found in all patients. A good correlation between the increment of DIC formation determined by analysis of 2,000 metaphases using Giemsa staining and those by 2,000 metaphases using Centromere-FISH was observed. However, no correlation was observed between the increment of DIC formation and the effective radiation dose. Therefore, these results suggest that chromosome cleavage may be induced by one CT scan, and we recommend 2,000 or more metaphases be analyzed in Giemsa staining or Centromere-FISH for DCAs in cases of low-dose radiation exposure.
Assuntos
Aberrações Cromossômicas/efeitos da radiação , Tomografia Computadorizada por Raios X/efeitos adversos , Idoso , Idoso de 80 Anos ou mais , Células Cultivadas , Feminino , Humanos , Hibridização in Situ Fluorescente , Linfócitos/metabolismo , Linfócitos/efeitos da radiação , Linfoma/diagnóstico por imagem , Linfoma/tratamento farmacológico , Linfoma/radioterapia , Masculino , Metáfase/genética , Metáfase/efeitos da radiação , Pessoa de Meia-Idade , Doses de Radiação , Radiação IonizanteRESUMO
Humans possess multiple specialized DNA polymerases that continue DNA replication beyond a variety of DNA lesions. DNA polymerase kappa (Pol κ) bypasses benzo[a]pyrene diolepoxide-N(2)-deoxyguanine (BPDE-N(2)-dG) DNA adducts in an almost error-free manner. In the previous work, we changed the amino acids close to the adducts in the active site and examined the bypass efficiency. The substitution of alanine for phenylalanine 171 (F171A) enhanced by 18-fold in vitro, the efficiencies of dCMP incorporation opposite (-)- and (+)-trans-anti-BPDE-N(2)-dG. In the present study, we established human cell lines that express wild-type Pol κ (POLK+/-), F171A (POLK F171A/-) or lack expression of Pol κ (POLK-/-) to examine the in vivo significance. These cell lines were generated with Nalm-6, a human pre-B acute lymphoblastic leukemia cell line, which has high efficiency for gene targeting. Mutations were analyzed with shuttle vectors having (-)- or (+)-trans-anti-BPDE-N(2)-dG in the supF gene. The frequencies of mutations were in the order of POLK-/->POLK+/->POLK F171A/- both in (-)- and (+)-trans-anti-BPDE-N(2)-dG. These results suggest that F171 may function as a molecular brake for bypass across BPDE-N(2)-dG by Pol κ and raise the possibility that the cognate substrates for Pol κ are not BP adducts in DNA but may be lesions in DNA induced by endogenous mutagens.
Assuntos
7,8-Di-Hidro-7,8-Di-Hidroxibenzo(a)pireno 9,10-óxido/análogos & derivados , Reparo do DNA , Replicação do DNA , DNA Polimerase Dirigida por DNA/genética , Desoxiguanosina/análogos & derivados , Substituição de Aminoácidos , Sequência de Bases , Domínio Catalítico , Linhagem Celular , Dano ao DNA , DNA Polimerase Dirigida por DNA/química , DNA Polimerase Dirigida por DNA/metabolismo , Desoxiguanosina/genética , Humanos , Mutagênese Sítio-Dirigida , Taxa de Mutação , Fenilalanina/genéticaRESUMO
DNA-protein crosslinks (DPCs)-where proteins are covalently trapped on the DNA strand-block the progression of replication and transcription machineries and hence hamper the faithful transfer of genetic information. However, the repair mechanism of DPCs remains largely elusive. Here we have analyzed the roles of nucleotide excision repair (NER) and homologous recombination (HR) in the repair of DPCs both in vitro and in vivo using a bacterial system. Several lines of biochemical and genetic evidence show that both NER and HR commit to the repair or tolerance of DPCs, but differentially. NER repairs DPCs with crosslinked proteins of sizes less than 12-14 kDa, whereas oversized DPCs are processed exclusively by RecBCD-dependent HR. These results highlight how NER and HR are coordinated when cells need to deal with unusually bulky DNA lesions such as DPCs.
Assuntos
Dano ao DNA , Reparo do DNA , Replicação do DNA , DNA/metabolismo , Recombinação Genética , Animais , Azacitidina/metabolismo , Cromossomos/genética , Reagentes de Ligações Cruzadas/metabolismo , DNA/genética , DNA Helicases/genética , DNA Helicases/metabolismo , Endodesoxirribonucleases/metabolismo , Escherichia coli/metabolismo , Proteínas de Escherichia coli/genética , Proteínas de Escherichia coli/metabolismo , Exodesoxirribonuclease V/genética , Exodesoxirribonuclease V/metabolismo , Formaldeído/metabolismo , Humanos , Plasmídeos/genética , Plasmídeos/metabolismoRESUMO
Nitric oxide and nitrous acid induce deamination of DNA bases, resulting in uracil, hypoxanthine, xanthine, and oxanine (Oxa) as major damage. Oxa reacts further with polyamines and DNA binding proteins, generating bulky cross-link adducts. Recently we have shown Oxa and cross-link adducts are potentially genotoxic lesions. In the present study, we have assessed the role of base excision repair (BER) and nucleotide excision repair (NER) systems in the repair of Oxa and Oxa-spermine (Oxa-Sp) cross-link adducts. Oxa was very poorly removed from DNA by both BER glycosylases and NER enzymes, whereas Oxa-Sp was efficiently excised by E. coli and human NER enzymes.
Assuntos
Adutos de DNA/metabolismo , Enzimas Reparadoras do DNA/metabolismo , Reparo do DNA , Nucleosídeos de Purina/metabolismo , Adutos de DNA/química , DNA Glicosilases/metabolismo , Humanos , Nucleosídeos de Purina/química , Espermina/análogos & derivados , Espermina/metabolismoRESUMO
5-Hydroxyuracil (HOU) and 5-hydroxycytosine (HOC) are major oxidative lesions of cytosine with mutagenic potentials. Therefore, HOU and HOC need to be removed from DNA to avoid mutation. In this study, oligonucleotide substrates containing HOU and HOC were synthesized by DNA polymerase reactions and tested for DNA glycosylases. Ung exhibited an extremely low activity for HOU as compared to uracil (U). In contrast, hSMUG1 excised HOU and U with a comparable efficiency. Ung and hSMUG1 did not excise HOC.
Assuntos
Citosina/metabolismo , Reparo do DNA , N-Glicosil Hidrolases/metabolismo , Estresse Oxidativo , Cromatografia de Afinidade , DNA GlicosilasesRESUMO
Endo III and Endo VIII are major E. coli DNA glycosylases that remove oxidatively damaged pyrimidine bases. In the present study, we have compared the damage specificity of human homologues of Endo III (hNTHl) and Endo VIII (hNEIL1 and hNEIL2) to elucidate the repair role in cells. hNTH1 and hNEIL1 recognized a similar spectra of bases lesions, but the preference of damage including the stereoisomers of thymine glycol was significantly different between hNTH1 and hNEIL1. hNEIL2 exhibited a strong AP lyase activity but the N-glycosylase activity for the tested oxidative base lesions was marginal.
Assuntos
Dano ao DNA , DNA Glicosilases/metabolismo , Pirimidinas/metabolismo , Células HeLa , Humanos , Oxirredução , Soluções , Estereoisomerismo , Especificidade por Substrato , Timina/análogos & derivados , Timina/químicaRESUMO
In human cells, oxidative pyrimidine lesions are restored by the base excision repair pathway initiated by homologues of Endo III (hNTH1) and Endo VIII (hNEIL1 and hNEIL2). In this study we have quantitatively analyzed and compared their activity toward nine oxidative base lesions and an apurinic/apyrimidinic (AP) site using defined oligonucleotide substrates. hNTH1 and hNEIL1 but not hNEIL2 excised the two stereoisomers of thymine glycol (5R-Tg and 5S-Tg), but their isomer specificity was markedly different: the relative activity for 5R-Tg:5S-Tg was 13:1 for hNTH1 and 1.5:1 for hNEIL1. This was also the case for their Escherichia coli homologues: the relative activity for 5R-Tg:5S-Tg was 1:2.5 for Endo III and 3.2:1 for Endo VIII. Among other tested lesions for hNTH1, an AP site was a significantly better substrate than urea, 5-hydroxyuracil (hoU), and guanine-derived formamidopyrimidine (mFapyG), whereas for hNEIL1 these base lesions and an AP site were comparable substrates. In contrast, hNEIL2 recognized an AP site exclusively, and the activity for hoU and mFapyG was marginal. hNEIL1, hNEIL2, and Endo VIII but not hNTH1 and Endo III formed cross-links to oxanine, suggesting conservation of the -fold of the active site of the Endo VIII homologues. The profiles of the excision of the Tg isomers with HeLa and E. coli cell extracts closely resembled those of hNTH1 and Endo III, confirming their major contribution to the repair of Tg isomers in cells. However, detailed analysis of the cellular activity suggests that hNEIL1 has a significant role in the repair of 5S-Tg in human cells.