RESUMO
XRCC1 protein is required for DNA single-strand break repair and genetic stability but its biochemical role is unknown. Here, we report that XRCC1 interacts with human polynucleotide kinase in addition to its established interactions with DNA polymerase-beta and DNA ligase III. Moreover, these four proteins are coassociated in multiprotein complexes in human cell extract and together they repair single-strand breaks typical of those induced by reactive oxygen species and ionizing radiation. Strikingly, XRCC1 stimulates the DNA kinase and DNA phosphatase activities of polynucleotide kinase at damaged DNA termini and thereby accelerates the overall repair reaction. These data identify a novel pathway for mammalian single-strand break repair and demonstrate a concerted role for XRCC1 and PNK in the initial step of processing damaged DNA ends.
Assuntos
Reparo do DNA/fisiologia , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Polinucleotídeo 5'-Hidroxiquinase/genética , Polinucleotídeo 5'-Hidroxiquinase/metabolismo , Animais , Células CHO , Cricetinae , Dano ao DNA/fisiologia , DNA Ligase Dependente de ATP , DNA Ligases/genética , DNA Ligases/metabolismo , DNA Polimerase beta/genética , DNA Polimerase beta/metabolismo , DNA de Cadeia Simples/genética , DNA de Cadeia Simples/metabolismo , Ativação Enzimática/genética , Humanos , Monoéster Fosfórico Hidrolases/genética , Monoéster Fosfórico Hidrolases/metabolismo , Proteínas de Ligação a Poli-ADP-Ribose , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Proteína 1 Complementadora Cruzada de Reparo de Raio-X , Proteínas de Xenopus , LevedurasRESUMO
Mammalian polynucleotide kinases catalyze the 5'-phosphorylation of nucleic acids and can have associated 3'-phosphatase activity, predictive of an important function in DNA repair following ionizing radiation or oxidative damage. The sequences of three tryptic peptides from a bovine 60-kDa polypeptide that correlated with 5'-DNA kinase and 3'-phosphatase activities identified human and murine dbEST clones. The 57.1-kDa conceptual translation product of this gene, polynucleotide kinase 3'-phosphatase (PNKP), contained a putative ATP binding site and a potential 3'-phosphatase domain with similarity to L-2-haloacid dehalogenases. BLAST searches identified possible homologs in Caenorhabditis elegans, Schizosaccharomyces pombe, and Drosophila melanogaster. The gene was localized to chromosome 19q13.3-13.4. Northern analysis indicated a 2-kilobase mRNA in eight human tissues. A glutathione S-transferase-PNKP fusion protein displayed 5'-DNA kinase and 3'-phosphatase activities. PNKP is the first gene for a DNA-specific kinase from any organism. PNKP expression partially rescued the sensitivity to oxidative damaging agents of the Escherichia coli DNA repair-deficient xth nfo double mutant. PNKP gene function restored termini suitable for DNA polymerase, consistent with in vivo removal of 3'-phosphate groups, facilitating DNA repair.
Assuntos
Dano ao DNA , Reparo do DNA , Monoéster Fosfórico Hidrolases/genética , Polinucleotídeo 5'-Hidroxiquinase/metabolismo , Sequência de Aminoácidos , Animais , Sequência de Bases , Southern Blotting , Bovinos , Mapeamento Cromossômico , Clonagem Molecular , DNA Complementar/química , Humanos , Peróxido de Hidrogênio/toxicidade , Dados de Sequência Molecular , Oxirredução , Monoéster Fosfórico Hidrolases/fisiologiaRESUMO
Mammalian polynucleotide kinases (PNKs) carry out 5'-phosphorylation of nucleic acids. Although the cellular function(s) of these enzymes remain to be delineated, important suggestions have included a role in DNA repair and, more recently, in DNA replication. Like T4 PNK, some preparations of mammalian PNKs have been reported to have an associated 3'-phosphatase activity. Previously, we have identified in calf thymus glands an apparently novel PNK with a neutral to alkaline pH optimum that lacked 3'-phosphatase activity. In this report, we describe purification of another bovine PNK, SNQI-PNK, with a slightly acidic pH optimum that copurifies with a 3'-phosphatase activity. The enzyme appears to be a monomer of 60 kDa. Mammalian DNA replication reactions were supplemented with T4 PNK or SNQI-PNK, and no significant effect on DNA replication in vitro was observed. Database searches support the earlier mapping of the 3'-phosphatase activity of T4 PNK to the C-terminus and suggest that the 3'-phosphatase domain of T4 PNK is related to the protein superfamily of L-2-haloacid dehalogenases. Exopeptidase digestion experiments were carried out to compare the SNQI-PNK enzyme with T4 PNK and led to the inference that the domain organization of the bovine polypeptide may differ from that of the T4 enzyme.
Assuntos
Bacteriófago T4/enzimologia , Replicação do DNA , Monoéster Fosfórico Hidrolases/química , Polinucleotídeo 5'-Hidroxiquinase/isolamento & purificação , Timo/enzimologia , Sequência de Aminoácidos , Animais , Bovinos , Cromatografia em Gel , Cromatografia por Troca Iônica , Eletroforese em Gel de Poliacrilamida , Células HeLa , Humanos , Dados de Sequência Molecular , Polinucleotídeo 5'-Hidroxiquinase/química , Polinucleotídeo 5'-Hidroxiquinase/metabolismo , Homologia de Sequência de AminoácidosRESUMO
Mutagenesis by the human bladder carcinogen 4-aminobiphenyl (ABP) was studied in single-stranded DNA from a bacteriophage M13 cloning vector. In comparison to ABP lesions in double-stranded DNA, lesions in single-stranded DNA were approximately 70-fold more mutagenic and 50-fold more genotoxic. Sequencing analysis of ABP-induced mutations in the lacZ gene revealed exclusively base-pair substitutions, with over 80% of the mutations occurring at G sites; the G at position 6310 accounted for 25% of the observed mutations. Among the sequence changes at G sites, G-->T transversions predominated, followed by G-->C transversions and G-->A transitions. In order to further elucidate the mutagenic mechanism of ABP, an oligonucleotide containing the major DNA adduct, N-(deoxyguanosin-8-yl)-4-aminobiphenyl (dG(8-ABP)), was situated within the PstI site of a single-stranded M13 genome. After in vivo replication of the adduct containing ABP-modified and control (unadducted) genomes, the mutational frequency and mutational specificity of the dG(8-ABP) lesion were determined. The targeted mutational efficiency was approximately 0.01%, and the primary mutation observed was the G-->C transversion. Thus dG(8-ABP), albeit weakly mutagenic at the PstI site, can contribute to the mutational spectrum of ABP lesions.
Assuntos
Compostos de Aminobifenil/química , Adutos de DNA/química , DNA de Cadeia Simples/genética , Nucleotídeos de Guanina/química , Mutagênese , Sequência de Aminoácidos , Bacteriófago M13 , Sequência de Bases , DNA Viral/química , Desoxirribonucleases de Sítio Específico do Tipo II , Dados de Sequência Molecular , Mutagênese Sítio-DirigidaRESUMO
Proteins that catalyze 5' phosphorylation of an oligodeoxyribonucleotide substrate can be fractionated by polymin P treatment of whole cell extracts of calf thymus glands. Anion exchange chromatography on Q-Sepharose revealed three separable peaks of activity in the polymin P supernatant fraction, and one peak of activity in the Polymin P pellet fraction. The latter activity, Polymin P-precipitable polynucleotide kinase (PP-PNK), was further purified with a 1,500-fold increase of specific activity compared to the crude Polymin P pellet fraction. Oligonucleotides, a dephosphorylated 2.9-kb EcoRI fragment, and poly(A) were phosphorylated by the enzyme preparation, but thymidine 3' monophosphate was not a substrate. PP-PNK preparations exhibited an apparent KM of 52 microM for ATP and 8 microM for oligo dT25. The enzyme preparation displayed no detectable 3' phosphatase or cyclic 2',3' phosphohydrolase activities. The sedimentation coefficient of the PP-PNK activity was 3.8S as determined by sucrose density gradient analysis; the Stokes radius was 45 A, leading to an estimated molecular mass of 72 kDa. The enzyme had a pH optimum in the neutral to alkaline range in several buffer systems and is distinct from the DNA kinase with an acidic pH optimum previously described in calf thymus.
Assuntos
DNA/metabolismo , Polietilenoimina/química , Polinucleotídeo 5'-Hidroxiquinase/isolamento & purificação , Timo/metabolismo , Animais , Bovinos , Cromatografia de Afinidade , Cromatografia por Troca Iônica , Feminino , Concentração de Íons de Hidrogênio , Técnicas In Vitro , Cinética , Masculino , Oligodesoxirribonucleotídeos/metabolismo , Fosforilação , Polinucleotídeo 5'-Hidroxiquinase/química , Sais/químicaRESUMO
Mammalian DNA ligase I has been shown to be a phosphoprotein. Dephosphorylation of purified DNA ligase I causes inactivation, an effect dependent on the presence of the N-terminal region of the protein. Expression of full-length human DNA ligase I in Escherichia coli yielded soluble but catalytically inactive enzyme whereas an N-terminally truncated form expressed activity. Incubation of the full-length preparation from E. coli with purified casein kinase II (CKII) resulted in phosphorylation of the N-terminal region and was accompanied by activation of the DNA ligase. Of a variety of purified protein kinases tested, only CKII stimulated the activity of calf thymus DNA ligase I. Tryptic phosphopeptide analysis of DNA ligase I revealed that CKII specifically phosphorylated a major peptide also apparently phosphorylated in cells, implying that CKII is a protein kinase acting on DNA ligase I in the cell nucleus. These data suggest that DNA ligase I is negatively regulated by its N-terminal region and that this inhibition can be relieved by post-translational modification.
Assuntos
DNA Ligases/metabolismo , Proteínas Serina-Treonina Quinases/metabolismo , Timo/enzimologia , Aminoácidos/análise , Animais , Sítios de Ligação , Caseína Quinase II , Bovinos , Linhagem Celular , Clonagem Molecular , DNA Ligase Dependente de ATP , DNA Ligases/genética , DNA Ligases/isolamento & purificação , Ativação Enzimática , Escherichia coli/genética , Humanos , Modelos Estruturais , Músculos/enzimologia , Fosfopeptídeos/isolamento & purificação , Fosforilação , Proteínas Serina-Treonina Quinases/isolamento & purificação , Coelhos , Proteínas Recombinantes/isolamento & purificação , Proteínas Recombinantes/metabolismoRESUMO
Human cDNA clones encoding the major DNA ligase activity in proliferating cells, DNA ligase I, were isolated by two independent methods. In one approach, a human cDNA library was screened by hybridization with oligonucleotides deduced from partial amino acid sequence of purified bovine DNA ligase I. In an alternative approach, a human cDNA library was screened for functional expression of a polypeptide able to complement a cdc9 temperature-sensitive DNA ligase mutant of Saccharomyces cerevisiae. The sequence of an apparently full-length cDNA encodes a 102-kDa protein, indistinguishable in size from authentic human DNA ligase I. The deduced amino acid sequence of the human DNA ligase I cDNA is 40% homologous to the smaller DNA ligases of S. cerevisiae and Schizosaccharomyces pombe, homology being confined to the carboxyl-terminal regions of the respective proteins. Hybridization between the cloned sequences and mRNA and genomic DNA indicates that the human enzyme is transcribed from a single-copy gene on chromosome 19.
Assuntos
DNA Ligases/genética , Polinucleotídeo Ligases/genética , Saccharomyces cerevisiae/genética , Sequência de Aminoácidos , Animais , Sequência de Bases , Clonagem Molecular , DNA Ligase Dependente de ATP , DNA Ligases/biossíntese , Teste de Complementação Genética , Humanos , Células Híbridas/enzimologia , Dados de Sequência Molecular , Sondas de Oligonucleotídeos , Proteínas Recombinantes/biossíntese , Homologia de Sequência do Ácido NucleicoRESUMO
Recent studies on eukaryotic DNA ligases are briefly reviewed. The two distinguishable enzymes from mammalian cells, DNA ligase I and DNA ligase II, have been purified to homogeneity and characterized biochemically. Two distinct DNA ligases have also been identified in Drosophila melanogaster embryos. The genes encoding DNA ligases from Schizosaccharomyces pombe, Saccharomyces cerevisiae and vaccinia virus have been cloned and sequenced. These 3 proteins exhibit about 30% amino acid sequence identity; the 2 yeast enzymes share 53% amino acid sequence identity or conserved changes. Altered DNA ligase I activity has been found in cell lines from patients with Bloom's syndrome, although a causal link between the enzyme deficiency and the disease has not yet been proven.
Assuntos
DNA Ligases/metabolismo , Polinucleotídeo Ligases/metabolismo , Sequência de Aminoácidos , Animais , Síndrome de Bloom/enzimologia , DNA Ligases/isolamento & purificação , DNA Ligases/fisiologia , Replicação do DNA , Humanos , Mamíferos , Dados de Sequência Molecular , Saccharomyces/enzimologia , Homologia de Sequência do Ácido Nucleico , Vaccinia virus/genéticaRESUMO
Mammalian DNA ligase I is presumed to act in DNA replication. Rabbit antibodies against the homogeneous enzyme from calf thymus inhibited DNA ligase I activity and consistently recognized a single polypeptide of 125 kDa when cells from an established bovine kidney cell line (MDBK) were lysed rapidly by a variety of procedures and subjected to immunoblotting analysis. After biosynthetic labeling of MDBK cells with [35S]methionine, immunoprecipitation experiments revealed a polypeptide of 125 kDa that did not appear when purified calf thymus DNA ligase I was used in competition. A 125-kDa polypeptide was adenylated when immunoprecipitated protein from MDBK cells was incubated with [alpha-32P]ATP. Thus, the apparent molecular mass of the initial translation product is identical or nearly so to that of the purified enzyme. The half-life of the protein is 7 h as determined by pulse-chase experiments in asynchronous MDBK cells. Immunocytochemistry and indirect immunofluorescence experiments showed that DNA ligase I is localized to cell nuclei.
Assuntos
DNA Ligases/metabolismo , Polinucleotídeo Ligases/metabolismo , Monofosfato de Adenosina/metabolismo , Animais , Western Blotting , Bovinos , Compartimento Celular , Núcleo Celular/enzimologia , Células Cultivadas/enzimologia , DNA Ligase Dependente de ATP , DNA Ligases/biossíntese , Imunofluorescência , Peso Molecular , Testes de PrecipitinaRESUMO
DNA ligase I is the major DNA ligase activity in proliferating mammalian cells. The protein has been purified to apparent homogeneity from calf thymus. It has a monomeric structure and a blocked N-terminal residue. DNA ligase I is a 125-kDa polypeptide as estimated by sodium dodecyl sulfate-gel electrophoresis and by gel chromatography under denaturing conditions, whereas hydrodynamic measurements indicate that the enzyme is an asymmetric 98-kDa protein. Immunoblotting with rabbit polyclonal antibodies to the enzyme revealed a single polypeptide of 125 kDa in freshly prepared crude cell extracts of calf thymus. Limited digestion of the purified DNA ligase I with several reagent proteolytic enzymes generated a relatively protease-resistant 85-kDa fragment. This domain retained full catalytic activity. Similar results were obtained with partially purified human DNA ligase I. The active large fragment represents the C-terminal part of the intact protein, and contains an epitope conserved between mammalian DNA ligase I and yeast and vaccinia virus DNA ligases. The function of the N-terminal region of DNA ligase I is unknown.
Assuntos
DNA Ligases/metabolismo , Polinucleotídeo Ligases/metabolismo , Animais , Sítios de Ligação , Western Blotting , Bovinos , Reações Cruzadas , DNA Ligase Dependente de ATP , DNA Ligases/imunologia , DNA Ligases/isolamento & purificação , Epitopos , Humanos , Peso Molecular , Mapeamento de Peptídeos , Subtilisinas/farmacologia , Timo/enzimologia , UltracentrifugaçãoAssuntos
Síndrome de Bloom/genética , DNA Ligases/deficiência , Polinucleotídeo Ligases/deficiência , Monofosfato de Adenosina/metabolismo , Animais , Síndrome de Bloom/enzimologia , Bovinos , Linhagem Celular , DNA/metabolismo , DNA Ligase Dependente de ATP , DNA Ligases/genética , DNA Ligases/isolamento & purificação , DNA Ligases/metabolismo , Replicação do DNA , Humanos , Linfócitos/enzimologia , Troca de Cromátide Irmã , Timo/enzimologiaRESUMO
Mutagenesis by N-acetoxy-N-trifluoroacetyl-4-aminobiphenyl, a reactive form of the human bladder carcinogen 4-aminobiphenyl (ABP), was studied in Escherichia coli virus M13mp10. N-acetoxy-N-trifluoroacetyl-4-ABP-treated DNA containing 140 lesions/duplex genome, when introduced into excision repair-competent cells induced for SOS mutagenic processing, resulted in a 40-fold increase in mutation frequency over background in the lacZ alpha gene fragment. DNA sequence changes were determined for 20 independent mutants. G-C base pairs were the major targets for base pair substitution mutations, although significant mutagenic activity was also observed at certain A-T base pairs. Deletion and frameshift mutations also were found in this sample. The salient feature of this partial "mutational spectrum" was a hotspot that occurred at position 6357 (amino acid 30 of the beta-galactosidase fragment encoded by M13mp10); this A-T to T-A transversion appeared in 6 of the 20 mutants. The property of ABP to mutate A-T base pairs was consistent with the result that N-hydroxy-ABP reverted Salmonella typhimurium strain TA104, which is presumed to revert primarily due to mutations at these sites. The ability of the major carcinogen-DNA adduct formed by ABP in vivo and in vitro, N-(deoxyguanosin-8-yl)-4-aminobiphenyl, to cause base pair substitution mutations was also investigated. This adduct was positioned specifically in the minus strand at position 6270 in duplex M13mp10 DNA. In the presence of the mutagenesis-enhancing plasmid pGW16 and UV induction of SOS mutagenic processing, it was shown that fewer than 0.02% of the adducts resulted in transition or transversion mutations following transfection of DNA into excision-repair competent cells. Similar results were obtained in uvrA and uvrC backgrounds. Although the major adduct did not cause base substitution mutations under these experimental conditions, the contribution of this lesion to the entire spectrum of mutations in the lacZ alpha fragment seems likely.
Assuntos
Compostos de Aminobifenil/toxicidade , Desoxiadenosinas/análogos & derivados , Neoplasias da Bexiga Urinária/induzido quimicamente , Sequência de Aminoácidos , Sequência de Bases , Desoxiadenosinas/toxicidade , Escherichia coli/efeitos dos fármacos , Humanos , Dados de Sequência Molecular , Testes de MutagenicidadeRESUMO
The duplex genome of Escherichia coli virus M13mp10 was modified at a unique site to contain N-(deoxyguanosin-8-yl)-4-aminobiphenyl (dG8-ABP), the major carcinogen-DNA adduct of the human bladder carcinogen 4-aminobiphenyl. A tetradeoxynucleotide containing a single dG8-ABP residue was synthesized by reacting 5'-d(TpGpCpA)-3' with N-acetoxy-N-(trifluoracetyl)-4-aminobiphenyl, followed by high-performance liquid chromatography purification of the principal reaction product 5'-d(TpG8-ABPpCpA)-3' (yield 15-30%). Characterization by fast atom bombardment mass spectrometry confirmed the structure as an intact 4-aminobiphenyl-modified tetranucleotide, while 1H nuclear magnetic resonance spectroscopy established the site of substitution and the existence of ring stacking between the carcinogen residue and DNA bases. Both 5'-d(TpG8-ABPpCpA)-3' and 5'-d(TpGpCpA)-3' were 5'-phosphorylated by use of bacteriophage T4 polynucleotide kinase and were incorporated into a four-base gap uniquely positioned in the center of the recognition site for the restriction endonuclease PstI, in an otherwise duplex genome of M13mp10. In the case of the adducted tetranucleotide, dG8-ABP was located in the minus strand at genome position 6270. Experiments in which the tetranucleotides were 5' end labeled with [32P]phosphate revealed the following: the adducted oligomer, when incubated in a 1000-fold molar excess in the presence of T4 DNA ligase and ATP, was found to be incorporated into the gapped DNA molecules with an efficiency of approximately 30%, as compared to the unadducted d(pTpGpCpA), which was incorporated with 60% ligation efficiency; radioactivity from the 5' end of each tetranucleotide was physically mapped to a restriction fragment that contained the PstI site and represented 0.2% of the genome; the presence of the lesion within the PstI recognition site inhibited the ability of PstI to cleave the genome at this site; in genomes in which ligation occurred, T4 DNA ligase was capable of covalently joining both modified and unmodified tetranucleotides to the gapped structures on both the 5' and the 3' ends with at least 90% efficiency. Evidence also is presented showing that the dG8-ABP-modified tetranucleotide was stable to the conditions of the recombinant DNA techniques used to insert it into the viral genome.(ABSTRACT TRUNCATED AT 400 WORDS)
Assuntos
Compostos de Aminobifenil , Compostos de Aminobifenil/farmacologia , Carcinógenos/farmacologia , Colífagos/genética , Desoxiguanosina/análogos & derivados , Escherichia coli/genética , Genes Virais , Mutagênicos , Compostos de Aminobifenil/análise , DNA Ligases/metabolismo , Replicação do DNA , Desoxiguanosina/análise , Desoxiguanosina/farmacologia , Indicadores e Reagentes , Espectroscopia de Ressonância Magnética , Espectrometria de Massas , Oligodesoxirribonucleotídeos/síntese químicaRESUMO
O6-Alkylguanine-DNA-alkyltransferase is a DNA repair protein known to carry out the transfer of alkyl groups from the O6-position of guanine in alkylated DNA to a cysteine acceptor site contained within its own protein sequence. We have examined the ability of this protein isolated from either E. coli or mammalian cells to perform this repair reaction in short oligodeoxynucleotides. Dodecadeoxynucleotides of the sequence 5'-dCGNGAATTCm6GCG-3' where N is any one of the normal four bases were all repaired very rapidly by the protein with 50% repair in less than 15 s at 0 degree C. The hexadeoxynucleotide 5'-dCGCm6GCG-3' was repaired slightly more slowly with 50% removal taking 7 min at 0 degree C and 1.5 min at 37 degrees C. The tetradeoxynucleotide 5'-dTm6GCA-3' was also a substrate but was repaired much more slowly requiring 45 min for 50% repair at 37 degrees C. These results indicate that (a) the AGT has a strong but not absolute preference for double-stranded DNA substrates; (b) the repair of O6-methylguanine is independent of the base opposite the lesion; and (c) that oligodeoxynucleotides as short as tetramers are substrates for repair by this protein.