RESUMEN

Control of tuberculosis depends both on an effective, accurate, and rapid diagnosis and an effective treatment and management. Antituberculous drugs have been used for more than 50 years and are likely ineffective against multidrug-resistant strains, leading to an urgent need for new drugs. Comparative genome analysis has indicated that Mycobacterium tuberculosis uvrC, a component of nucleotide excision repair (NER) system, is an essential gene without any human homolog. This raises the possibility to use this gene as a new drug target. This study investigated the essential role of uvrC on growth of M. tuberculosis in the presence of DNA damaging agents, UV light and hydrogen peroxide (generator of reactive oxygen species). Results revealed that the M. tuberculosis uvrC mutant was more sensitive to UV than the control strain (p < 0.01), but was not more sensitive to hydrogen peroxide. These results showed that uvrC is essential for M. tuberculosis DNA repair system, particularly in response to DNA damage caused by UV irradiation.

Asunto(s)

Daño del ADN , Reparación del ADN , Endodesoxirribonucleasas/genética , Endodesoxirribonucleasas/efectos de la radiación , Genes Bacterianos , Genes Esenciales , Mycobacterium tuberculosis/genética , Mycobacterium tuberculosis/efectos de la radiación , Rayos Ultravioleta/efectos adversos , Análisis de Varianza , Cartilla de ADN , Silenciador del Gen , Humanos , Peróxido de Hidrógeno/farmacología , Estrés Oxidativo

RESUMEN

DNA mismatch repair (MMR) and very-short patch (VSP) repair are two pathways involved in the repair of T:G mismatches. To learn about competition and cooperation between these two repair pathways, we analyzed the physical and functional interaction between MutL and Vsr using biophysical and biochemical methods. Analytical ultracentrifugation reveals a nucleotide-dependent interaction between Vsr and the N-terminal domain of MutL. Using chemical crosslinking, we mapped the interaction site of MutL for Vsr to a region between the N-terminal domains similar to that described before for the interaction between MutL and the strand discrimination endonuclease MutH of the MMR system. Competition between MutH and Vsr for binding to MutL resulted in inhibition of the mismatch-provoked MutS- and MutL-dependent activation of MutH, which explains the mutagenic effect of Vsr overexpression. Cooperation between MMR and VSP repair was demonstrated by the stimulation of the Vsr endonuclease in a MutS-, MutL- and ATP-hydrolysis-dependent manner, in agreement with the enhancement of VSP repair by MutS and MutL in vivo. These data suggest a mobile MutS-MutL complex in MMR signalling, that leaves the DNA mismatch prior to, or at the time of, activation of downstream effector molecules such as Vsr or MutH.

Asunto(s)

Adenosina Trifosfatasas/metabolismo , Reparación de la Incompatibilidad de ADN , Endodesoxirribonucleasas/metabolismo , Proteínas de Escherichia coli/metabolismo , Adenosina Trifosfatasas/química , Adenosina Trifosfatasas/efectos de la radiación , Reactivos de Enlaces Cruzados , Enzimas Reparadoras del ADN/metabolismo , Proteínas de Unión al ADN/metabolismo , Endodesoxirribonucleasas/química , Endodesoxirribonucleasas/efectos de la radiación , Proteínas de Escherichia coli/química , Proteínas de Escherichia coli/efectos de la radiación , Proteínas MutL , Proteína MutS de Unión a los Apareamientos Incorrectos del ADN/metabolismo , Procesos Fotoquímicos , Estructura Terciaria de Proteína , Ultracentrifugación

RESUMEN

Alpha-anomeric 2'-deoxynucleosides (alphadN) are one of the products formed by ionizing radiation (IR) in DNA under anoxic conditions. Alpha-2'-deoxyadenosine (alphadA) and alpha-thymidine (alphaT) are not recognized by DNA glycosylases, and are likely removed by the alternative nucleotide incision repair (NIR) pathway. Indeed, it has been shown that alphadA is a substrate for the Escherichia coli Nfo and human Ape1 proteins. However, the repair pathway for removal of alphadA and other alphadN in yeast is unknown. Here we report that alphadA when present in DNA is recognized by the Saccharomyces cerevisiae Apn1 protein, a homologue of Nfo. Furthermore, alphaT is a substrate for Nfo and Apn1. Kinetic constants indicate that alphadA and alphaT are equally good substrates, as a tetrahydrofuranyl (THF) residue, for Nfo and Apn1. Using E. coli and S. cerevisiae cell-free extracts, we have further substantiated the role of the nfo and apn1 gene products in the repair of alphadN. Surprisingly, we found that bacteria and yeast NIR-deficient mutants are not sensitive to IR, suggesting that DNA strand breaks with terminal 3'-blocking groups rather than alphadN might contribute to cell survival. We propose that the novel substrate specificities of Nfo and Apn1 play an important role in counteracting oxidative DNA base damage.

Asunto(s)

Daño del ADN , Reparación del ADN , ADN-(Sitio Apurínico o Apirimidínico) Liasa/química , Desoxiadenosinas/química , Desoxirribonucleasa IV (Fago T4-Inducido)/metabolismo , Endodesoxirribonucleasas/metabolismo , Proteínas de Escherichia coli/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Anaerobiosis/genética , Anaerobiosis/efectos de la radiación , Emparejamiento Base , Sistema Libre de Células/metabolismo , Sistema Libre de Células/efectos de la radiación , Reparación del ADN/efectos de la radiación , Enzimas Reparadoras del ADN , ADN-(Sitio Apurínico o Apirimidínico) Liasa/efectos de la radiación , Desoxiadenosinas/efectos de la radiación , Desoxirribonucleasa IV (Fago T4-Inducido)/genética , Desoxirribonucleasa IV (Fago T4-Inducido)/efectos de la radiación , Endodesoxirribonucleasas/genética , Endodesoxirribonucleasas/efectos de la radiación , Escherichia coli/enzimología , Escherichia coli/genética , Escherichia coli/efectos de la radiación , Proteínas de Escherichia coli/genética , Proteínas de Escherichia coli/efectos de la radiación , Rayos gamma , Humanos , Cinética , Mutagénesis , Ácidos Nucleicos Heterodúplex/química , Ácidos Nucleicos Heterodúplex/genética , Ácidos Nucleicos Heterodúplex/efectos de la radiación , Saccharomyces cerevisiae/enzimología , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/efectos de la radiación , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/efectos de la radiación , Especificidad por Sustrato/genética , Especificidad por Sustrato/efectos de la radiación , Timidina/metabolismo , Timidina/efectos de la radiación

RESUMEN

3-Aminobenzamide (3AB) is an inhibitor of poly (ADP-ribose) polymerase (PARP), an enzyme implicated in the maintenance of genomic integrity, which is activated in response to radiation-induced DNA strand breaks. cDNA macroarray membranes containing 1536 clones were used to characterize the gene expression profiles displayed by mouse BALB/3T3 fibroblasts (A31 cell line) in response to ionizing irradiation alone or in combination with 3AB. A31 cells in exponential growth were pre-treated with 3AB 4mM 1h before gamma-irradiation (4Gy), remaining in culture during 6h until harvesting time. A31 cells treated with 3AB alone presented a down-regulation in genes involved in protein processing and cell cycle control, while an up-regulation of genes involved in apoptosis and related to DNA/RNA synthesis and repair was verified. A31 cells irradiated with 4Gy displayed 41 genes differentially expressed, being detected a down-regulation of genes involved in protein processing and apoptosis, and genes controlling the cell cycle. Concomitantly, another set of genes for protein processing and related to DNA/RNA synthesis and repair were found to be up-regulated. A positive or negative interaction effect between 3AB and radiation was verified for 29 known genes. While the combined treatment induced a synergistic effect on the expression of LCK proto-oncogene and several genes related to protein synthesis/processing, a negative interaction effect was found for the expression of genes related to cytoskeleton and extracellular matrix assembly (SATB1 and Anexin III), cell cycle control (tyrosine kinase), and genes participating in DNA/RNA synthesis and repair (RNA helicase, FLAP endonuclease-1, DNA-3 glycosylase methyladenine, splicing factor SC35 and Soh1). The present data open the possibility to investigate the direct participation of specific genes, or gene products acting in concert in the mechanism underlying the cell response to radiation-induced DNA damage under the influence of PARP inhibitor.

Asunto(s)

Células 3T3/efectos de la radiación , Benzamidas/farmacología , Inhibidores Enzimáticos/farmacología , Regulación de la Expresión Génica/efectos de la radiación , Ribonucleoproteínas , Células 3T3/efectos de los fármacos , Células 3T3/fisiología , Animales , Anexina A3/efectos de los fármacos , Anexina A3/genética , Anexina A3/efectos de la radiación , Daño del ADN/genética , Daño del ADN/efectos de la radiación , Endodesoxirribonucleasas/efectos de los fármacos , Endodesoxirribonucleasas/genética , Endodesoxirribonucleasas/efectos de la radiación , Endonucleasas de ADN Solapado , Rayos gamma , Perfilación de la Expresión Génica , Regulación de la Expresión Génica/efectos de los fármacos , Proteína Tirosina Quinasa p56(lck) Específica de Linfocito/efectos de los fármacos , Proteína Tirosina Quinasa p56(lck) Específica de Linfocito/genética , Proteína Tirosina Quinasa p56(lck) Específica de Linfocito/efectos de la radiación , Proteínas de Unión a la Región de Fijación a la Matriz/efectos de los fármacos , Proteínas de Unión a la Región de Fijación a la Matriz/genética , Proteínas de Unión a la Región de Fijación a la Matriz/efectos de la radiación , Ratones , Proteínas Nucleares/efectos de los fármacos , Proteínas Nucleares/genética , Proteínas Nucleares/efectos de la radiación , Análisis de Secuencia por Matrices de Oligonucleótidos , Inhibidores de Poli(ADP-Ribosa) Polimerasas , Proto-Oncogenes Mas , ARN/biosíntesis , ARN/efectos de los fármacos , ARN/efectos de la radiación , ARN Helicasas/efectos de los fármacos , ARN Helicasas/genética , ARN Helicasas/efectos de la radiación , Factores de Empalme Serina-Arginina

RESUMEN

The RusA protein of Escherichia coli is a DNA structure-specific endonuclease that resolves Holliday junction intermediates formed during DNA replication, recombination and repair by introducing symmetrically paired incisions 5' to CC dinucleotides. It is encoded by the defective prophage DLP12, which raises the possibility that it may be of bacteriophage origin. We show that rusA-like sequences are indeed often associated with prophage sequences in the genomes of several bacterial species. They are also found in many bacteriophages, including Lactococcus lactis phage r1t. However, rusA is also present in the chromosome of the hyperthermophilic bacterium Aquifex aeolicus. In this case, there is no obvious association of rusA with prophage-like sequences. Given the ancient lineage of Aquifex aeolicus, this observation provides the first indication that RusA may be of bacterial origin. The RusA proteins of A. aeolicus and bacteriophage r1t were purified and shown to resolve Holliday junctions. The r1t enzyme also promotes DNA repair in strains lacking the RuvABC resolvase. Both enzymes cleave junctions in a sequence-dependent manner, but the A. aeolicus RusA shows a different sequence preference (3' to TG) from the E. coli protein (5' to CC), and the r1t RusA has relaxed sequence dependence, requiring only a single cytosine.

Asunto(s)

Bacterias/genética , Bacteriófagos/genética , Endodesoxirribonucleasas/genética , Proteínas de Escherichia coli , Resolvasas de Unión Holliday , Lactococcus/virología , Secuencia de Aminoácidos , Secuencia de Bases , ADN Bacteriano/química , ADN Viral/química , Endodesoxirribonucleasas/química , Endodesoxirribonucleasas/efectos de la radiación , Cinética , Datos de Secuencia Molecular , Conformación de Ácido Nucleico , Mapeo Restrictivo , Alineación de Secuencia , Homología de Secuencia de Aminoácido

RESUMEN

Ultraviolet (UV) irradiation induces predominantly cyclobutane and (6-4) pyrimidine dimer photoproducts in DNA. Several mechanisms for repairing these mutagenic UV-induced DNA lesions have been identified. Nucleotide excision repair is a major pathway, but mechanisms involving photolyases and DNA glycosylases have also been characterized. Recently, a novel UV damage endonuclease (UVDE) was identified that initiates an excision repair pathway different from previously established repair mechanisms. Homologues of UVDE have been found in eukaryotes as well as in bacteria. In this report, we have used oligonucleotide substrates containing site-specific cyclobutane pyrimidine dimers and (6-4) photoproducts for the characterization of this UV damage repair pathway. After introduction of single-strand breaks at the 5' sides of the photolesions by UVDE, these intermediates became substrates for cleavage by flap endonucleases (FEN-1 proteins). FEN-1 homologues from humans, Saccharomyces cerevisiae, and Schizosaccharomyces pombe all cleaved the UVDE-nicked substrates at similar positions 3' to the photolesions. T4 endonuclease V-incised DNA was processed in the same way. Both nicked and flapped DNA substrates with photolesions (the latter may be intermediates in DNA polymerase-catalyzed strand displacement synthesis) were cleaved by FEN-1. The data suggest that the two enzymatic activities, UVDE and FEN-1, are part of an alternative excision repair pathway for repair of UV photoproducts.

Asunto(s)

Daño del ADN , Reparación del ADN , Endodesoxirribonucleasas/efectos de la radiación , Secuencia de Bases , Cartilla de ADN , Endodesoxirribonucleasas/genética , Endonucleasas de ADN Solapado , Reacción en Cadena de la Polimerasa , Dímeros de Pirimidina , Rayos Ultravioleta

RESUMEN

Changes in the level of blood lymphocyte chromatin degradation and relaxation were investigated after the whole body irradiation with doses up to 0.21 C/kg. It was shown that ionizing irradiation initiated the increase of nuclear endogenous Ca/mg-dependent endonuclease, and protease activities.

Asunto(s)

Cromatina/efectos de la radiación , Linfocitos/efectos de la radiación , Animales , Fraccionamiento Celular , Cromatina/química , Endodesoxirribonucleasas/efectos de la radiación , Endopeptidasas/efectos de la radiación , Linfocitos/química , Ratas , Ratas Wistar , Irradiación Corporal Total

RESUMEN

HeLa cells arrested in G2 phase 22 h after receiving 11.5 Gy gamma-radiation contained 3.6-fold more EDTA-resistant DNA repair endonuclease activity than unirradiated cells. Enzyme activity was determined by measuring the release of fragments from an irradiated repetitive alpha DNA substrate or from synthetic substrates containing a single modified base, 8-oxoguanine (8-oxo-G), a major radiation product. It appeared that the radiation-induced enhanced repair activity in some cells might be a feature of radiation-induced G2 arrest. Indeed, unirradiated G2 HeLa cells that had been synchronized by double thymidine block contained 3-7-fold more endonuclease activity than G1 or S-phase cells. Similarly, two of four other cell lines tested exhibited elevated repair endonuclease activity in G2. However, all six cell lines tested exhibited radiation-enhanced repair endonuclease activity. Therefore, the underlying mechanism for radiation enhancement of enzyme activity remains to be clarified and does not seem to be completely accounted for as a consequence of G2 arrest. The results showed different substrate specificities among cell lines as well as differences during the cell cycle of individual cell lines. Repair endonuclease activity from all cell lines which we have tested were associated with 60-70 kDa proteins from Superose 12 columns. Since reports from other laboratories have described several different DNA repair activities in 50-70 kDa Superose 12 fractions, it seems possible that the DNA repair enzymes may be associated in a repairosome structure.

Asunto(s)

Endodesoxirribonucleasas/metabolismo , Endodesoxirribonucleasas/efectos de la radiación , Fase G2/fisiología , Fase G2/efectos de la radiación , Animales , Secuencia de Bases , Ciclo Celular/fisiología , Ciclo Celular/efectos de la radiación , Línea Celular , ADN/metabolismo , ADN/efectos de la radiación , Daño del ADN , Reparación del ADN , ADN-(Sitio Apurínico o Apirimidínico) Liasa , Desoxirribonucleasa IV (Fago T4-Inducido) , Guanina/análogos & derivados , Guanina/metabolismo , Células HeLa , Humanos , Ratones , Datos de Secuencia Molecular , Oligonucleótidos/metabolismo

RESUMEN

Alkaline elution has been used for quantitative detection of DNA damage caused by ionizing radiation in unlabeled somatic and germ cells. Both the induction and subsequent repair have been studied for two classes of DNA damage, viz. single-strand breaks (SSB), and base damage (BD) recognized by the gamma-endonuclease activity in a cell-free extract of Micrococcus luteus bacteria. The high sensitivity of the assay permitted the measurement of induction and repair of SSB and BD after in vitro exposure of hamster germ cells in different cellular stages of spermatogenesis (spermatocytes, round and elongated spermatids), and of bone-marrow cells, to biologically relevant doses (0-8 Gy) of 60Co gamma-rays. A dose-dependent increase was observed for both types of lesions, which was similar for most cell types. The elongated spermatids, however, showed a lower induction frequency of SSB (and perhaps BD). Spermatocytes, round spermatids and bone-marrow cells had normal, fast repair of the SSB when compared with the repair reported for cultured rodent cells and human lymphocytes. In contrast, the elongated spermatids showed hardly any SSB repair. The initial rate of repair of BD in spermatocytes and bone-marrow cells was in the same range as that for SSB, but only 60-70% of the initial BD was repaired within 1 h, whereas after that period no SSB were detectable. The round spermatids hardly repaired any BD within the first hour after irradiation, but after 7 h only a few BD could be detected. In elongated spermatids repair of BD could not be measured due to a high background level of this type of damage.

Asunto(s)

Daño del ADN , Reparación del ADN , ADN de Cadena Simple/efectos de la radiación , Espermatogénesis/genética , Animales , Cricetinae , Endodesoxirribonucleasas/efectos de la radiación , Rayos gamma , Masculino , Mesocricetus , Espermatocitos/efectos de la radiación , Espermatogénesis/efectos de la radiación

RESUMEN

A general mechanism by which proteins locate their target sites within large domains of DNA is a one-dimensional facilitated diffusion process in which the protein scans DNA in a nonspecifically bound state. An electrostatic contribution to this type of mechanism has been previously established. This study was designed to question whether other characteristics of a protein's structure might contribute to the scanning mechanism of target site location. In this regard, T4 endonuclease V was shown to establish an ionic strength dependent monomer-dimer equilibrium in solution. A protein dimer interaction site was postulated to exist along a putative alpha-helix containing amino acid residues 54-62. The conservative substitutions of Phe-60----Leu-60 and Phe-59, Phe-60----Leu-59, Leu-60 resulted in mutant enzymes which remained in the monomeric state independent of the ionic strength of the solution. The target site location mechanism of these mutants has also been altered. Under conditions where wild-type endonuclease V processively scans nontarget DNA, the target location mechanism of the monomeric mutant proteins was shifted toward a less processive search. This decrease in the processivity of the mutants was especially surprising because the nontarget DNA binding affinity was found to be significantly increased. Thus, an additional component of the endonuclease V DNA scanning mechanism appears to be the formation of a stable endonuclease V dimer complex.

Asunto(s)

Endodesoxirribonucleasas/genética , Mutagénesis Sitio-Dirigida , Fagos T/enzimología , Proteínas Virales , Secuencia de Bases , Unión Competitiva , Reparación del ADN/efectos de la radiación , Desoxirribonucleasa (Dímero de Pirimidina) , Endodesoxirribonucleasas/efectos de la radiación , Escherichia coli/efectos de la radiación , Genes Bacterianos/efectos de la radiación , Datos de Secuencia Molecular , Conformación Proteica , Ingeniería de Proteínas , Dímeros de Pirimidina/genética , Fagos T/genética

RESUMEN

The denV gene of bacteriophage T4, which encodes the pyrimidine dimer-specific repair enzyme endonuclease V, was introduced into murine fibroblasts with normal rodent pyrimidine dimer repair capabilities. Endonuclease V recognizes ultraviolet radiation (UVR)-induced pyrimidine dimers and produces single-strand breaks adjacent to the dimers. These nicks may serve as substrates to initiate excision repair of pyrimidine dimers by endogenous enzymes. In the present study, murine fibroblasts stably transfected with denV were able to remove 50-80% of UVR-induced pyrimidine dimers, while control cells removed only about 20% of dimers under the same conditions of pyrimidine dimer induction and repair. For both control and denV-transfected cells, repair continued for at least 24 h after exposure. When removal of UVR-induced photoproducts was initiated by endogenous excision repair mechanisms, an average of 38 nucleotides were replaced per dimer removed, as determined by bromouracil photolysis; denV-initiated excision repair, on the other hand, resulted in removal of an average of 6 nucleotides per dimer repaired. The enhanced pyrimidine dimer repair capabilities conferred by denV gene expression did not appear to improve post-UVR survival.

Asunto(s)

Reparación del ADN/genética , Endodesoxirribonucleasas/metabolismo , Dímeros de Pirimidina/metabolismo , Animales , Southern Blotting , Bromouracilo/metabolismo , Línea Celular Transformada , Supervivencia Celular/genética , Supervivencia Celular/efectos de la radiación , Reparación del ADN/fisiología , Reparación del ADN/efectos de la radiación , ADN de Cadena Simple/metabolismo , Desoxirribonucleasa (Dímero de Pirimidina) , Endodesoxirribonucleasas/genética , Endodesoxirribonucleasas/efectos de la radiación , Fibroblastos , Cinética , Ratones , Plásmidos/genética , Fagos T/genética , Transfección/genética

RESUMEN

Nucleotide excision repair in Escherichia coli is initiated by (A)BC excinuclease, an enzyme which incises DNA on both sides of bulky adducts and removes the damaged nucleotide as a 12-13 base long oligomer. The incision pattern of the enzyme was examined using DNA modified by 4-nitroquinoline 1-oxide (4NQO) and UV light. Similar to the cleavage pattern of UV photoproducts and other bulky adducts, the enzyme incises the 8th phosphodiester bond 5' and 5th phosphodiester bond 3' to the 4NQO-modifed base, primarily guanine. The extent of DNA damage by these agents was determined using techniques which quantitatively cleave the DNA or stop at the site of the adduct. By comparison of the intensity of gel bands created by (A)BC excinuclease and the specific cleavage at the damaged site, the efficiency of (A)BC excinuclease incision at 13 different 4NQO-induced adducts and 13 different photoproducts was determined by densitometric scanning. In general, incisions made at 4NQO-induced adducts are proportional to the extent of damage, though the efficiency of cutting throughout the sequence tested varies from 25 to 75%. Incisions made at pyrimidine dimers are less efficient than at 4NQO-adducts, ranging from 13 to 65% incision relative to modification, though most are around 50%. The two (6-4) photoproducts within the region tested are incised more efficiently than any pyrimidine dimer.

Asunto(s)

4-Nitroquinolina-1-Óxido/toxicidad , Daño del ADN , ADN Bacteriano/efectos de la radiación , Endodesoxirribonucleasas/genética , Proteínas de Escherichia coli , Secuencia de Bases , Reparación del ADN , Endodesoxirribonucleasas/efectos de la radiación , Escherichia coli/enzimología , Datos de Secuencia Molecular , Rayos Ultravioleta

RESUMEN

The murine ascites sarcoma 180 cells were used to test the in vivo effectiveness of mitomycin C (MMC) and gamma-radiation applied in combination. The action of intraperitoneal administration of MMC and/or whole-body gamma irradiation on sarcoma 180 tumor bearing Swiss albino mice was investigated by studying the template activity of isolated tumor chromatin. The Km value for transcription of 10 Gy-irradiated chromatin was found to decrease with time implying an increase in the template efficiency with respect to that of the unirradiated control. Maximum decrease in Km was observed after 24 h of irradiation. MMC treatment (7 mg/kg body weight of mouse) for 18 h resulted in an inhibition of the transcription rate. Severe inhibition in the template activity was found when cells were subjected to MMC treatment 18 h prior to irradiation with 10 Gy. Susceptibility of tumor chromatin to DNase II followed the same pattern as observed in the case of transcription indicating structural alteration of the treated chromatin. The data showed that DNA damage and its consequences produced in the ascites cells by prior treatment of MMC were not repaired during the 18 h period after which the application of radiation enhanced cytotoxicity.

Asunto(s)

Cromatina/aislamiento & purificación , Mitomicinas/farmacología , Sarcoma 180/genética , Moldes Genéticos , Transcripción Genética , Animales , División Celular/efectos de los fármacos , División Celular/efectos de la radiación , Línea Celular , Terapia Combinada , Daño del ADN , Endodesoxirribonucleasas/efectos de la radiación , Cinética , Ratones , Mitomicina , Mitomicinas/uso terapéutico , Sarcoma 180/tratamiento farmacológico , Sarcoma 180/radioterapia , Moldes Genéticos/efectos de los fármacos , Moldes Genéticos/efectos de la radiación , Factores de Tiempo

RESUMEN

A cytoplasmic thymocyte fraction isolated 1 h after irradiation of mice accelerates chromatin degradation in isolated nuclei. Treatment of the cytoplasmic fraction by heat and injection of cycloheximide to mice prevent the acceleration of DNA degradation. The analysis of the chromatin degradation products and the kinetics of this process at acid and alkaline pH shows that activation of DNA degradation in thymocytes by a factor obtained from the irradiated cell cytoplasm is specific for a Ca2+, Mg2+-dependent enzyme. The time- and dose-dependent parameters of the appearance in the thymocyte cytoplasm of the factor influencing degradation of chromatin are in a good agreement with both the time of the onset of its postirradiation degradation and the dose dependence of this process.

Asunto(s)

Cromatina/efectos de los fármacos , Endodesoxirribonucleasas/efectos de la radiación , Timo/efectos de la radiación , Animales , Núcleo Celular/efectos de los fármacos , Núcleo Celular/metabolismo , Supervivencia Celular/efectos de los fármacos , Supervivencia Celular/efectos de la radiación , Cromatina/metabolismo , Cicloheximida/farmacología , Citoplasma/efectos de los fármacos , Citoplasma/enzimología , Citoplasma/efectos de la radiación , ADN/efectos de los fármacos , ADN/metabolismo , Relación Dosis-Respuesta en la Radiación , Endodesoxirribonucleasas/metabolismo , Activación Enzimática/efectos de los fármacos , Activación Enzimática/efectos de la radiación , Rayos gamma , Interfase/efectos de los fármacos , Interfase/efectos de la radiación , Ratones , Ratones Endogámicos C57BL , Ratones Endogámicos CBA , Timo/efectos de los fármacos , Timo/enzimología , Factores de Tiempo

RESUMEN

The properties of a DNA-repair endonuclease isolated from mouse plasmacytoma cells have been further studied. It acted on ultraviolet-light-irradiated supercoiled DNA, and the requirement for a supercoiled substrate was absolute at ultraviolet light doses below 1.5 kJ m-2. At higher doses relaxed DNA could also serve as a substrate, but the activity on this DNA was due mostly to hydrolysis of ultraviolet-light-induced apurinic/apyrimidinic (AP) sites by the AP-endonuclease activity associated with the enzyme. The latter enzyme activity did not require a supercoiled form of the DNA. The enzyme also introduced nicks in unirradiated d(A-T)n. The nicked ultraviolet-light-irradiated DNA served as a substrate for DNA polymerase I, showing that the nicks contained free 3'-OH ends. Treatment of the nicked ultraviolet-light-irradiated DNA with bacterial alkaline phosphatase followed by T4 polynucleotide kinase, resulted in the phosphorylation of the 5' ends of the nicks, indicating that the nicks possessed a 5'-phosphate group; 5'- and 3'-mononucleotide analyses of the labelled DNA suggested that the enzyme introduced breaks primarily between G and T residues. The enzyme did not act on any specific region on the supercoiled DNA molecule; it produced random nicks in ultraviolet-light-modified phi X 174 replicative form I DNA. Antibodies raised against ultraviolet-light-irradiated DNA inhibited the activity. DNA adducts such as N-acetoxy-2-acetylaminofluorene and psoralen were not recognized by the enzyme. It is suggested that the enzyme has a specificity directed toward helical distortions.

Asunto(s)

Endodesoxirribonucleasas/aislamiento & purificación , Plasmacitoma/enzimología , Animales , Sitios de Unión , Línea Celular , Reparación del ADN , ADN de Neoplasias/metabolismo , ADN de Neoplasias/efectos de la radiación , ADN Superhelicoidal/aislamiento & purificación , ADN-(Sitio Apurínico o Apirimidínico) Liasa , ADN Polimerasa Dirigida por ADN/aislamiento & purificación , ADN Polimerasa Dirigida por ADN/efectos de la radiación , Desoxirribonucleasa IV (Fago T4-Inducido) , Relación Dosis-Respuesta en la Radiación , Endodesoxirribonucleasas/efectos de la radiación , Ratones , Fosforilación , Rayos Ultravioleta

RESUMEN

The nuclei from the control and irradiated (3 h after irradiation at a dose of 10 Gy) thymocytes were preincubated with NAD in conditions optimal for poly (ADP) ribosylation. This was shown to decrease by 6-7- and 2-3 times, respectively, the rate of autolytic cleavage of DNA by Ca/Mg-dependent endonuclease. The inhibitors of poly (ADP-riboso)-polymerase, nicotine amide and thymidine, removed the effect of NAD. The data obtained prompt an assumption that the post-irradiation activation of Ca/Mg-nuclease in thymocytes is associated with the disturbance of its post-translation modification, poly(ADP)ribosylation.

Asunto(s)

Endodesoxirribonucleasas/metabolismo , Azúcares de Nucleósido Difosfato/metabolismo , Poli Adenosina Difosfato Ribosa/metabolismo , Procesamiento Proteico-Postraduccional , Animales , Núcleo Celular/enzimología , Núcleo Celular/efectos de la radiación , ADN/metabolismo , Endodesoxirribonucleasas/efectos de la radiación , Activación Enzimática/efectos de los fármacos , Activación Enzimática/efectos de la radiación , Rayos gamma , Masculino , NAD/metabolismo , Poli Adenosina Difosfato Ribosa/efectos de la radiación , Procesamiento Proteico-Postraduccional/efectos de los fármacos , Procesamiento Proteico-Postraduccional/efectos de la radiación , Ratas , Ratas Endogámicas , Timo/enzimología , Timo/efectos de la radiación

Asunto(s)

Reparación del ADN/efectos de la radiación , Replicación del ADN/efectos de la radiación , ADN/efectos de la radiación , Colifagos/efectos de la radiación , ADN Viral/efectos de la radiación , ADN-(Sitio Apurínico o Apirimidínico) Liasa , Desoxirribonucleasa IV (Fago T4-Inducido) , Relación Dosis-Respuesta en la Radiación , Endodesoxirribonucleasas/efectos de la radiación , Genes Virales/efectos de la radiación , Técnicas para Inmunoenzimas , Cinética , Radiación Ionizante , Especificidad por Sustrato , Timina/análogos & derivados , Timina/análisis

Asunto(s)

Cromatina/enzimología , Endodesoxirribonucleasas/metabolismo , Timo/ultraestructura , Animales , Cromatina/efectos de la radiación , ADN/metabolismo , ADN/efectos de la radiación , ADN-(Sitio Apurínico o Apirimidínico) Liasa , Desoxirribonucleasa IV (Fago T4-Inducido) , Endodesoxirribonucleasas/efectos de la radiación , Rayos gamma , Dosis de Radiación , Ratas , Timo/efectos de la radiación