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1.
Int J Radiat Biol ; 90(1): 53-9, 2014 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-24164476

RESUMO

PURPOSE: To test the hypothesis that differences in DNA double-strand breaks (DSB) repair fidelity underlies differences in radiosensitivity. MATERIALS AND METHODS: A primary fibroblast culture (C42) derived from a pediatric cancer patient treated with reduced radiation doses consequent to a family history of radiosensitivity reminiscent of chromosomal fragility syndrome, was compared to a normal control (C29). DNA DSB rejoining and repair fidelity were studied by Southern blotting and hybridization to specific fragments: Alu repetitive sequence representing the overall DSB rejoining capacity in the genome and a 3.2 Mbp NotI restriction fragment on chromosome 21 for DSB repair fidelity. RESULTS: Although both assays showed statistically significant difference (p ≤ 0.05) between the two cell strains in residual misrepaired (un-or mis-rejoined) DSB (24 h after 30 or 80 Gy), the residual damage was lower in the Alu enriched genome assay compared to NotI assay (0.01-0.07 and 0.10-0.37, respectively). CONCLUSIONS: These results suggest that, in comparison to classic DSB repair experiment, an assay of measuring DNA DSB repair fidelity can provide better resolution and a more accurate estimate of misrepair of radiation-induced DNA damage, which underlies genomic instability and increased radiosensitivity.


Assuntos
Transtornos Cromossômicos/genética , Fragilidade Cromossômica/genética , Fragilidade Cromossômica/efeitos da radiação , Dano ao DNA/genética , Reparo de Erro de Pareamento de DNA/genética , Reparo de Erro de Pareamento de DNA/efeitos da radiação , Tolerância a Radiação/genética , Pareamento Incorreto de Bases/genética , Pareamento Incorreto de Bases/efeitos da radiação , Bioensaio/métodos , Pré-Escolar , Feminino , Humanos
2.
Chem Soc Rev ; 40(12): 5718-29, 2011 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-21691619

RESUMO

Hybridization of DNA is one of the most intriguing events in molecular recognition and is essential for living matter to inherit life beyond generations. In addition to the function of DNA as genetic material, DNA hybridization is a key to control the function of DNA-based materials in nanoscience. Since the hybridization of two single stranded DNAs is a thermodynamically favorable process, dissociation of the once formed DNA duplex is normally unattainable under isothermal conditions. As the progress of DNA-based nanoscience, methodology to control the DNA hybridization process has become increasingly important. Besides many reports using the chemically modified DNA for the regulation of hybridization, we focused our attention on the use of a small ligand as the molecular glue for the DNA. In 2001, we reported the first designed molecule that strongly and specifically bound to the mismatched base pairs in double stranded DNA. Further studies on the mismatch binding molecules provided us a key discovery of a novel mode of the binding of a mismatch binding ligand that induced the base flipping. With these findings we proposed the concept of molecular glue for DNA for the unidirectional control of DNA hybridization and, eventually photoswitchable molecular glue for DNA, which enabled the bidirectional control of hybridization under photoirradiation. In this tutorial review, we describe in detail how we integrated the mismatch binding ligand into photoswitchable molecular glue for DNA, and the application and perspective in DNA-based nanoscience.


Assuntos
DNA/química , Luz , Hibridização de Ácido Nucleico/efeitos dos fármacos , Hibridização de Ácido Nucleico/efeitos da radiação , Pareamento Incorreto de Bases/efeitos dos fármacos , Pareamento Incorreto de Bases/efeitos da radiação , Sequência de Bases , DNA/genética , Naftiridinas/química , Temperatura
3.
Radiat Res ; 173(1): 98-109, 2010 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-20041764

RESUMO

Auger electron emitters like (125)I are the radionuclides of choice for gene-targeted radiotherapy. The highly localized damage they induce in DNA is produced by three mechanisms: direct damage by the emitted Auger electrons, indirect damage by diffusible free radicals produced by Auger electrons traveling in water, and charge neutralization of the residual, highly positively charged tellurium daughter atom by stripping electrons from covalent bonds of neighboring residues. The purpose of our work was to determine whether these mechanisms proceed through an intermediate energy transfer step along DNA. It was proposed that this intermediate step proceeds through the charge transport mechanism in DNA. Conventional charge transport has been described as either a hopping mechanism initiated by charge injection into DNA and propagated by charge migration along the DNA or a tunneling mechanism in which charge moves directly from a donor to an acceptor within DNA. Well-known barriers for the hopping mechanism were used to probe the role of charge transport in (125)I-induced DNA damage. We studied their effect on the distribution of DNA breaks produced by the decay of (125)I in samples frozen at -80 degrees C. We found that these barriers had no measurable effect on the distribution of (125)I-induced breaks.


Assuntos
Dano ao DNA , Radioisótopos do Iodo/efeitos adversos , Pareamento Incorreto de Bases/efeitos dos fármacos , Pareamento Incorreto de Bases/efeitos da radiação , Sequência de Bases , Bromodesoxiuridina/farmacologia , DNA/genética , DNA/metabolismo , Transporte de Elétrons/efeitos dos fármacos , Guanina/análogos & derivados , Guanina/farmacologia
4.
DNA Repair (Amst) ; 8(9): 1055-67, 2009 Sep 02.
Artigo em Inglês | MEDLINE | ID: mdl-19497792

RESUMO

In response to genomic insults cells trigger a signal transduction pathway, known as DNA damage checkpoint, whose role is to help the cell to cope with the damage by coordinating cell cycle progression, DNA replication and DNA repair mechanisms. Accumulating evidence suggests that activation of the first checkpoint kinase in the cascade is not due to the lesion itself, but it requires recognition and initial processing of the lesion by a specific repair mechanism. Repair enzymes likely convert a variety of physically and chemically different lesions to a unique common structure, a ssDNA region, which is the checkpoint triggering signal. Checkpoint kinases can modify the activity of repair mechanisms, allowing for efficient repair, on one side, and modulating the generation of the ssDNA signal, on the other. This strategy may be important to allow the most effective repair and a prompt recovery from the damage condition. Interestingly, at least in some cases, if the damage level is low enough the cell can deal with the lesions and it does not need to activate the checkpoint response. On the other hand if damage level is high or if the lesions are not rapidly repairable, checkpoint mechanisms become important for cell survival and preservation of genome integrity.


Assuntos
Ciclo Celular , Dano ao DNA , Reparo do DNA , Animais , Pareamento Incorreto de Bases/efeitos da radiação , Ciclo Celular/efeitos da radiação , DNA/biossíntese , Reparo do DNA/efeitos da radiação , Humanos , Raios Ultravioleta
5.
J Bacteriol ; 191(2): 555-62, 2009 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-19011038

RESUMO

In this study, we investigated the role of the nucleotide excision repair (NER) pathway in mycobacterial DNA repair. Mycobacterium smegmatis lacking the NER excinuclease component uvrB or the helicase uvrD1 gene and a double knockout lacking both genes were constructed, and their sensitivities to a series of DNA-damaging agents were analyzed. As anticipated, the mycobacterial NER system was shown to be involved in the processing of bulky DNA adducts and interstrand cross-links. In addition, it could be shown to exert a protective effect against oxidizing and nitrosating agents. Interestingly, inactivation of uvrB and uvrD1 significantly increased marker integration frequencies in gene conversion assays. This implies that in mycobacteria (which lack the postreplicative mismatch repair system) NER, and particularly the UvrD1 helicase, is involved in the processing of a subset of recombination-associated mismatches.


Assuntos
Proteínas de Bactérias/metabolismo , DNA Helicases/metabolismo , Reparo do DNA , Mycobacterium smegmatis/enzimologia , Mycobacterium smegmatis/genética , Proteínas de Bactérias/genética , Pareamento Incorreto de Bases/efeitos da radiação , DNA Helicases/genética , Reparo do DNA/efeitos da radiação , Conversão Gênica/efeitos da radiação , Mutação/efeitos da radiação , Mycobacterium smegmatis/efeitos da radiação , Raios Ultravioleta
6.
Mol Pharmacol ; 74(3): 863-71, 2008 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-18535288

RESUMO

The antitumor drug 5-fluoro-2'-deoxyuridine (FdUrd) also sensitizes tumor cells to ionizing radiation in vitro and in vivo. Although radiosensitization with FdUrd requires dTTP depletion and S-phase arrest, the exact mechanism by which these events produce radiosensitization remains unknown. We hypothesized that the depletion of dTTP produces DNA mismatches that, if not repaired before irradiation, would result in radiosensitization. We evaluated this hypothesis in mismatch repair (MMR)-deficient HCT116 0-1 cells that lack the expression of the required MMR protein MLH1 (inactive MLH1), and in MMR-proficient (wild-type MLH1) HCT116 1-2 cells. Although HCT116 0-1 cells were less sensitive to FdUrd (IC(50) = 3.5 microM) versus HCT116 1-2 cells (IC(50) = 0.75 microM), when irradiation followed FdUrd (IC(50)) the MLH1-inactivated cells exhibited greater radiosensitization compared with MMR-wild-type cells [radiation enhancement ratio (RER) = 1.8 +/- 0.28 versus 1.1 +/- 0.1, respectively] and an increase (> or =8-fold) in nucleotide misincorporations. In SW620 cells and HCT116 1-2 MLH1-wild-type cells, FdUrd (IC(50)) did not produce radiosensitization nor did it increase the mutation frequency, but after short hairpin RNA-directed suppression of MLH1 this concentration produced excellent radiosensitization (RER = 1.6 +/- 0.10 and 1.5 +/- 0.06, respectively) and an increase in nucleotide misincorporations (8-fold and 6-fold, respectively). Incubation with higher concentrations of FdUrd (IC(90)) after suppression of MLH1 produced a further increase in ionizing radiation sensitivity in both SW620 and HCT116 1-2 cells (RER = 1.8 +/- 0.03 and 1.7 +/- 0.13, respectively) and nucleotide misincorporations (>10-fold in both cell lines). These results demonstrate an important role for MLH1 and implicate mismatches in radiosensitization by FdUrd.


Assuntos
Proteínas Adaptadoras de Transdução de Sinal/deficiência , Pareamento Incorreto de Bases/efeitos dos fármacos , Floxuridina/farmacologia , Proteínas Nucleares/deficiência , Tolerância a Radiação/efeitos dos fármacos , Pareamento Incorreto de Bases/efeitos da radiação , Ciclo Celular/efeitos dos fármacos , Ciclo Celular/efeitos da radiação , Morte Celular/efeitos dos fármacos , Morte Celular/efeitos da radiação , Células HCT116 , Humanos , Proteína 1 Homóloga a MutL , Mutação/genética , Nucleotídeos/metabolismo , Plasmídeos/genética , RNA Interferente Pequeno/metabolismo , Tolerância a Radiação/efeitos da radiação , Radiação Ionizante
7.
Mol Cell Biol ; 28(4): 1373-82, 2008 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-18086882

RESUMO

DNA polymerases play a central role during homologous recombination (HR), but the identity of the enzyme(s) implicated remains elusive. The pol3-ct allele of the gene encoding the catalytic subunit of DNA polymerase delta (Poldelta) has highlighted a role for this polymerase in meiotic HR. We now address the ubiquitous role of Poldelta during HR in somatic cells. We find that pol3-ct affects gene conversion tract length during mitotic recombination whether the event is initiated by single-strand gaps following UV irradiation or by site-specific double-strand breaks. We show that the pol3-ct effects on gene conversion are completely independent of mismatch repair, indicating that shorter gene conversion tracts in pol3-ct correspond to shorter extensions of primed DNA synthesis. Interestingly, we find that shorter repair tracts do not favor synthesis-dependent strand annealing at the expense of double-strand-break repair. Finally, we show that the DNA polymerases that have been previously suspected to mediate HR repair synthesis (Polepsilon and Poleta) do not affect gene conversion during induced HR, including in the pol3-ct background. Our results argue strongly for the preferential recruitment of Poldelta during HR.


Assuntos
DNA Polimerase III/metabolismo , DNA Fúngico/metabolismo , Ácidos Nucleicos Heteroduplexes/metabolismo , Recombinação Genética , Saccharomyces cerevisiae/enzimologia , Pareamento Incorreto de Bases/efeitos da radiação , Troca Genética/efeitos da radiação , Quebras de DNA de Cadeia Dupla/efeitos da radiação , DNA Polimerase II/metabolismo , Reparo do DNA/efeitos da radiação , Desoxirribonucleases de Sítio Específico do Tipo II/metabolismo , Homozigoto , Perda de Heterozigosidade/efeitos da radiação , Viabilidade Microbiana/efeitos da radiação , Mitose/efeitos da radiação , Modelos Genéticos , Proteína 2 Homóloga a MutS/metabolismo , Polimorfismo de Fragmento de Restrição , Radiação Ionizante , Recombinação Genética/efeitos da radiação , Saccharomyces cerevisiae/citologia , Saccharomyces cerevisiae/efeitos da radiação , Proteínas de Saccharomyces cerevisiae/metabolismo
8.
Rev Sci Instrum ; 78(8): 085111, 2007 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-17764359

RESUMO

We have developed a novel apparatus that allows us to irradiate nonvolatile organic films of high mass (1-100 microg range) spread out over a large surface area (42 cm(2)) with low energy (kT-100 eV) heavy ions and to quantitatively analyze the film substance via standard biochemical techniques afterwards. Here we discuss the details of the apparatus and method and show that it allows us to measure substantial damage to double stranded DNA molecules (plasmids) and its fundamental subunits induced by heavy ions with unprecedented low energies, i.e., 2.5 eV/amu; these energies correspond to track end energies of stopping ions or secondary ions created along primary ion tracks. We find that hyperthermal Ar(+) ions interacting with plasmid DNA will lead to the formation of single and double strand breaks, as well as fragmentation of nucleosides, which also involve chemical modifications and site specific rupture along the N1-C1 glycosidic bond, resulting in base release. In cells, such localized clustered damage will enhance the severity of DNA strand lesions, thus making them harder to repair.


Assuntos
Dano ao DNA , DNA/química , DNA/efeitos da radiação , Íons Pesados , Aceleradores de Partículas/instrumentação , Pareamento Incorreto de Bases/efeitos da radiação , Desenho Assistido por Computador , DNA/genética , Quebras de DNA , Fragmentação do DNA/efeitos da radiação , Relação Dose-Resposta à Radiação , Desenho de Equipamento , Análise de Falha de Equipamento , Doses de Radiação , Reprodutibilidade dos Testes , Sensibilidade e Especificidade
9.
Radiat Prot Dosimetry ; 122(1-4): 136-40, 2006.
Artigo em Inglês | MEDLINE | ID: mdl-17185311

RESUMO

This paper presents results of (125)I effects on plasmid pBR322 in aqueous solution, simulating the complete transport of Auger and X rays up to the chemical phase. In addition to new sampling algorithms, new electronic cross sections are included. Simulations were carried out both with (125)I, bound to plasmid, or free, in its vicinity. The influence of the hydroxyl radical scavenger dimethyl sulfoxyde (DMSO) has also been tested, underlying that, in naked DNA, double strand breaks (caused by the decay of bound (125)I) are mainly due to direct hits. The calculated yields of relaxation events (RE) and linearization events (LE) show good agreement with experimental ones: when (125)I is bound to the plasmid pBR322, 0.16 RE and 0.83 LE per decay (without DMSO) are then observed. Then, when 2 mol DMSO is added, RE and LE probabilities become 0.22 and 0.76. The very light differences with those from literature could arise from experimental conditions.


Assuntos
Pareamento Incorreto de Bases/efeitos da radiação , Dano ao DNA , Radioisótopos do Iodo/química , Modelos Químicos , Plasmídeos/química , Plasmídeos/efeitos da radiação , Simulação por Computador , Relação Dose-Resposta à Radiação , Meia-Vida , Transferência Linear de Energia , Modelos Moleculares , Doses de Radiação , Radiação Ionizante
10.
Radiat Prot Dosimetry ; 122(1-4): 169-72, 2006.
Artigo em Inglês | MEDLINE | ID: mdl-17145725

RESUMO

Although DNA damage is widely viewed as a critical target for the induction of cell killing by ionising radiation, the exact nature of DNA damage responsible for these effects is unknown. To address this issue, the probability of forming lethal damage by single proton tracks, derived from published survival data for Chinese hamster V79 cells irradiated by protons with energies from 0.57 to 5.01 MeV, has been compared to estimated yields of clustered DNA lesions and repair outcomes calculated with Monte Carlo models. The reported studies provide new information about the potential relationship between the induction and repair of clustered DNA damage and trends in the expected number of lethal events for protons with increasing linear energy transfer (LET). A good correlation was found between the number of lethal events in V79 cells and the induction of double-strand breaks (DSBs) consisting of three or more elementary DNA lesions. For the yields of other types of DNA damage, as well as point mutations formed through the misrepair of base damage and single-strand breaks, observed trends with increasing LET are not consistent with trends in the yields of lethal events. This observation suggests that the relative biological effectiveness (RBE) of protons of varying quality may be more closely related to the induction of complex DSBs rather than other forms of damage.


Assuntos
Dano ao DNA , DNA/genética , DNA/efeitos da radiação , Fibroblastos/fisiologia , Fibroblastos/efeitos da radiação , Modelos Biológicos , Radiação Ionizante , Animais , Pareamento Incorreto de Bases/efeitos da radiação , Biofísica/métodos , Sobrevivência Celular/efeitos da radiação , Cricetinae , Cricetulus , Relação Dose-Resposta à Radiação , Fibroblastos/citologia , Transferência Linear de Energia/fisiologia , Transferência Linear de Energia/efeitos da radiação , Doses de Radiação
11.
Radiat Prot Dosimetry ; 122(1-4): 163-5, 2006.
Artigo em Inglês | MEDLINE | ID: mdl-17132657

RESUMO

We present here evidence showing that the yields of DNA lesions induced by He(2+) ions strongly depend on Linear energy transfer (LET). In this study, hydrated plasmid DNA was irradiated with He(2+) ions with LET values of 19, 63 and 95 keVmicrom(-1). The yields of prompt single-strand breaks (SSBs) are very similar at the varying LET values, whereas the yields of prompt double-strand breaks (DSBs) increase with increasing LET. Further, base lesions were revealed as additional strand breaks by post-irradiation treatment of the DNA with endonuclease III (Nth) and formamidopyrimidine-DNA glycosylase (Fpg). The reduction in the yield of these enzymatically induced SSBs and DSBs becomes significant as the LET increases. These results suggest that the clustering of DNA lesions becomes more probable in regions of high LET.


Assuntos
Dano ao DNA , DNA Bacteriano/química , DNA Bacteriano/efeitos da radiação , Hélio , Modelos Químicos , Radioisótopos , Pareamento Incorreto de Bases/efeitos da radiação , Simulação por Computador , DNA Bacteriano/genética , Relação Dose-Resposta à Radiação , Íons , Transferência Linear de Energia , Modelos Moleculares , Doses de Radiação , Radiação Ionizante
12.
Radiat Prot Dosimetry ; 122(1-4): 86-8, 2006.
Artigo em Inglês | MEDLINE | ID: mdl-17132664

RESUMO

The yields of soft-X-ray-induced DNA damages have been measured by using closed-circular plasmid DNA. Several DNA solutions with three kinds of radical scavenger capacity and also fully hydrated DNA samples were irradiated to compare the contribution by indirect reaction of diffusible water radicals, such as OH*, with those by direct action of secondary electrons. The yields of prompt single- (SSBs) and double-strand breaks (DSBs) decrease with increasing scavenging capacity. The SSB yields for soft X-rays are approximately midway those between gamma-ray and ultrasoft X-ray data previously reported. Heat labile sites are observed only in the low scavenger condition. The yields of the base lesions revealed by post irradiation treatment with base excision repair enzymes showed a similar value for Nth and Fpg protein except in the hydrated sample. These results indicate that the direct effect of soft X-rays induces the damages with different efficiency from those by indirect effect.


Assuntos
Pareamento Incorreto de Bases/efeitos da radiação , Dano ao DNA , Modelos Químicos , Plasmídeos/química , Plasmídeos/efeitos da radiação , Raios X , Simulação por Computador , Relação Dose-Resposta à Radiação , Doses de Radiação
13.
Biochemistry ; 44(6): 1932-40, 2005 Feb 15.
Artigo em Inglês | MEDLINE | ID: mdl-15697218

RESUMO

Interstrand cross-links (ICL) represent one of the most toxic types of DNA damage for dividing cells. They are induced both by natural products (e.g., psoralens + UVA) and by several chemical agents, some of which are used in chemotherapy (e.g., carboplatin and mitomycin C). Although repair mechanisms exist for interstrand cross-links, these lesions can induce mutations, chromosomal rearrangements, and cell death. Here, we report, for the first time, the formation of ICL by gamma-rays in brominated DNA. It is well established that the radiosensitization properties of bromodeoxyuridine (BrdUrd) result primarily from the electrophilic nature of the bromine, making it a good leaving group and leading to the irreversible formation of a uridinyl radical (dUrd(*)) or uridinyl anion (dUrd-) upon addition of an electron. We observe that the radiolytic loss of the bromine atom is greatly suppressed in double-stranded compared to single-stranded DNA. We have used a model DNA containing a bulge, formed by five mismatched bases, and have observed a linear dose-response for the formation of strand breaks on the single-stranded regions of both the brominated strand and the opposite nonbrominated strand. Surprisingly, we have observed the formation of interstrand cross-links exclusively in the mismatched region. Thus, we propose that the radiosensitization effects of bromodeoxyuridine in vivo will almost certainly be limited to single strand regions such as found in transcription bubbles, replication forks, mismatched DNA, and possibly the loop region of telomeres. Our results suggest that interstrand cross-links may contribute to the radiosensitization effects of BrdUrd. These findings may have profound implications for the clinical use of bromodeoxyuridine as a radiosensitizer, as well as for the development of targeted radiosensitizers.


Assuntos
Bromodesoxiuridina/toxicidade , Dano ao DNA , DNA/toxicidade , Raios gama , Radiossensibilizantes/toxicidade , Pareamento Incorreto de Bases/efeitos da radiação , Bromo/efeitos da radiação , Bromodesoxiuridina/metabolismo , Quebra Cromossômica , DNA/metabolismo , DNA/efeitos da radiação , DNA Complementar/metabolismo , DNA Complementar/efeitos da radiação , DNA de Cadeia Simples/metabolismo , DNA de Cadeia Simples/efeitos da radiação , Ácidos Nucleicos Heteroduplexes/metabolismo , Ácidos Nucleicos Heteroduplexes/efeitos da radiação , Hibridização de Ácido Nucleico/efeitos da radiação , Oligonucleotídeos/metabolismo , Oligonucleotídeos/efeitos da radiação , Radiossensibilizantes/metabolismo
14.
J Radiat Res ; 45(2): 229-37, 2004 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-15304965

RESUMO

Ionizing radiation generates diverse DNA lesions that differentially induce cell death and mutations. In the present study, calf thymus DNA (400 microg/ml) and HeLa cells were irradiated by (60)Co gamma-rays, and abasic (AP) sites and endonuclease (Endo)III- and 8-oxoguanine glycosylase (hOGG1)-sensitive base modifications in DNA were quantitated by the aldehyde reactive probe (ARP) assay. The irradiation of calf thymus DNA in phosphate buffer generated 91 Endo III- and 100 hOGG1-sensitive base modifications and 110 AP sites per 10(6) base pairs (bp) per Gy. The yield of the lesions in Tris buffer was 41- to 91-fold lower than that in phosphate, demonstrating a radioprotective effect of Tris. The HeLa cell chromosomal DNA contained 12 Endo III- and 3.8 hOGG1-sensitive base modifications and less than 1 AP sites per 10(6) bp as endogenous damage, and their level was increased by irradiation. The yields of the damage at 1 Gy (roughly equivalent to the lethal dose of HeLa cells [1.6-1.8 Gy]) were 0.13 Endo III, 0.091 hOGG1, and 0.065 AP sites per 10(6) bp, showing that irradiation with a lethal dose brought about only a marginal increase in base damage relative to an endogenous one. A comparison of the present data with those reported for DNA strand breaks supports the primary importance of double-strand breaks and clustered lesions as lethal damages formed by ionizing radiation.


Assuntos
Biotina/análogos & derivados , Dano ao DNA , Análise Mutacional de DNA/métodos , DNA-Formamidopirimidina Glicosilase/química , DNA/química , DNA/efeitos da radiação , Desoxirribonuclease (Dímero de Pirimidina)/química , Proteínas de Escherichia coli/química , Pareamento Incorreto de Bases/efeitos da radiação , DNA/metabolismo , DNA-Formamidopirimidina Glicosilase/metabolismo , Desoxirribonuclease (Dímero de Pirimidina)/metabolismo , Relação Dose-Resposta à Radiação , Proteínas de Escherichia coli/metabolismo , Células HeLa , Humanos , Doses de Radiação
15.
Photochem Photobiol ; 79(5): 461-9, 2004 May.
Artigo em Inglês | MEDLINE | ID: mdl-15191056

RESUMO

Allele-specific polymerase chain reaction is based on polymerase extension from primers that contain a 3' end base that is complementary to a specific mutation and inhibition of extension with wild-type DNA due to a 3' end mismatch. Taq polymerase is commonly used for this assay, but because of the high rate of nucleotide extension from primer 3' base mismatches documented for Taq polymerase, high sensitivity is difficult to achieve. To determine whether other polymerases might improve assay sensitivity, 15 polymerases were tested with mutation-specific primers for two ultraviolet-induced mutations in the human 5S ribosomal RNA genes. Of the 15 polymerases tested, six were capable of discriminating these mutations at levels equivalent to or better than Taq polymerase. All primers were phosphorothioate modified on the 3' end to block removal of the critical 3' mutation-specific base by polymerases containing 3' --> 5' exonuclease "proofreading" activity. The effectiveness of phosphorothioate modification was measured in mock polymerase chain reaction reactions and a time course. All six enzymes containing this exonuclease activity showed some ability to digest phosphorothioate-modified primers and could be divided into two groups, showing fast and slow digestion kinetics. Of the three enzymes that showed slow digestion kinetics, two also showed significantly slower digestion kinetics of unmodified primers.


Assuntos
Análise Mutacional de DNA/métodos , Primers do DNA/química , DNA Polimerase Dirigida por DNA/metabolismo , Reação em Cadeia da Polimerase/métodos , Tionucleotídeos/química , Raios Ultravioleta , Alelos , Pareamento Incorreto de Bases/genética , Pareamento Incorreto de Bases/efeitos da radiação , Sequência de Bases , Primers do DNA/metabolismo , Células HeLa , Humanos , Dados de Sequência Molecular , RNA Ribossômico 5S/genética , Taq Polimerase/metabolismo , Tionucleotídeos/metabolismo
16.
Nucleic Acids Symp Ser (Oxf) ; (48): 101-2, 2004.
Artigo em Inglês | MEDLINE | ID: mdl-17150498

RESUMO

We examined the effect of silver (I) cation on the thermal stability of heteroduplex and homoduplex. Addition of silver (I) cation increased the melting temperature of heteroduplex containing C:C mismatch base pair by about 3-4 degrees C. The thermal stability of homoduplex and heteroduplexes containing other kinds of mismatch base pairs was not significantly changed by the addition of silver (I) cation. We conclude that silver (I) cation specifically stabilizes heteroduplex containing C:C mismatch base pair. Our results certainly support the idea that the addition of silver (I) cation to C:C mismatch base pair in heteroduplex could be a convenient strategy for heteroduplex analysis and may eventually lead to progress in single nucleotide polymorphism genotyping.


Assuntos
Pareamento Incorreto de Bases/efeitos dos fármacos , Ácidos Nucleicos Heteroduplexes/efeitos dos fármacos , Polimorfismo de Nucleotídeo Único/genética , Prata/farmacologia , Pareamento Incorreto de Bases/efeitos da radiação , Sequência de Bases , Concentração de Íons de Hidrogênio , Dados de Sequência Molecular , Desnaturação de Ácido Nucleico/efeitos dos fármacos , Desnaturação de Ácido Nucleico/efeitos da radiação , Ácidos Nucleicos Heteroduplexes/genética , Ácidos Nucleicos Heteroduplexes/efeitos da radiação , Temperatura de Transição , Raios Ultravioleta
17.
Nucleic Acids Res ; 28(20): 3887-96, 2000 Oct 15.
Artigo em Inglês | MEDLINE | ID: mdl-11024167

RESUMO

Poly(ADP-ribose) polymerase 1 (PARP-1) is a nuclear enzyme that is activated by binding to DNA breaks induced by ionizing radiation or through repair of altered bases in DNA by base excision repair. Mice lacking PARP-1 and, in certain cases, the cells derived from these mice exhibit hypersensitivity to ionizing radiation and alkylating agents. In this study we investigated base excision repair in cells lacking PARP-1 in order to elucidate whether their augmented sensitivity to DNA damaging agents is due to an impairment of the base excision repair pathway. Extracts prepared from wild-type cells or cells lacking PARP-1 were similar in their ability to repair plasmid DNA damaged by either X-rays (single-strand DNA breaks) or by N:-methyl-N:'-nitro-N:-nitrosoguanidine (methylated bases). In addition, we demonstrated in vivo that PARP-1-deficient cells treated with N:-methyl-N:'-nitro-N:-nitrosoguanidine repaired their genomic DNA as efficiently as wild-type cells. Therefore, we conclude that cells lacking PARP-1 have a normal capacity to repair single-strand DNA breaks inflicted by X-irradiation or breaks formed during the repair of modified bases. We propose that the hypersensitivity of PARP-1 null mutant cells to gamma-irradiation and alkylating agents is not directly due to a defect in DNA repair itself, but rather results from greatly reduced poly(ADP-ribose) formation during base excision repair in these cells.


Assuntos
Pareamento Incorreto de Bases/genética , Reparo do DNA/genética , Deleção de Genes , Poli(ADP-Ribose) Polimerases/deficiência , Animais , Pareamento Incorreto de Bases/efeitos dos fármacos , Pareamento Incorreto de Bases/efeitos da radiação , Extratos Celulares , Linhagem Celular , Sistema Livre de Células , Dano ao DNA/efeitos dos fármacos , Dano ao DNA/genética , Dano ao DNA/efeitos da radiação , Metilação de DNA/efeitos dos fármacos , Reparo do DNA/efeitos dos fármacos , Reparo do DNA/efeitos da radiação , DNA de Cadeia Simples/biossíntese , DNA de Cadeia Simples/efeitos dos fármacos , DNA de Cadeia Simples/genética , DNA de Cadeia Simples/efeitos da radiação , Relação Dose-Resposta à Radiação , Fibroblastos , Cinética , Metilnitronitrosoguanidina/farmacologia , Camundongos , Modelos Genéticos , Mutagênicos/farmacologia , NAD/metabolismo , Plasmídeos/efeitos dos fármacos , Plasmídeos/genética , Plasmídeos/efeitos da radiação , Poli(ADP-Ribose) Polimerases/genética , Poli(ADP-Ribose) Polimerases/fisiologia , Tolerância a Radiação , Raios X
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