RESUMO
Oxidatively damaged DNA results from the attack of sugar and base moieties by reactive oxygen species (ROS), which are formed as byproducts of normal cell metabolism and during exposure to endogenous or exogenous chemical or physical agents. Guanine, having the lowest redox potential, is the DNA base the most susceptible to oxidation, yielding products such as 8-oxo-7,8-dihydroguanine (8-oxoG) and 2-6-diamino-4-hydroxy-5-formamidopyrimidine (FapyG). In DNA, 8-oxoG was shown to be mutagenic yielding GC to TA transversions upon incorporation of dAMP opposite this lesion by replicative DNA polymerases. In prokaryotic and eukaryotic cells, 8-oxoG is primarily repaired by the base excision repair pathway (BER) initiated by a DNA N-glycosylase, Fpg and OGG1, respectively. In Escherichia coli, Fpg cooperates with MutY and MutT to prevent 8-oxoG-induced mutations, the "GO-repair system". In Saccharomyces cerevisiae, OGG1 cooperates with nucleotide excision repair (NER), mismatch repair (MMR), post-replication repair (PRR) and DNA polymerase η to prevent mutagenesis. Human and mouse cells mobilize all these pathways using OGG1, MUTYH (MutY-homolog also known as MYH), MTH1 (MutT-homolog also known as NUDT1), NER, MMR, NEILs and DNA polymerases η and λ, to prevent 8-oxoG-induced mutations. In fact, mice deficient in both OGG1 and MUTYH develop cancer in different organs at adult age, which points to the critical impact of 8-oxoG repair on genetic stability in mammals. In this review, we will focus on Fpg and OGG1 proteins, their biochemical and structural properties as well as their biological roles. Other DNA N-glycosylases able to release 8-oxoG from damaged DNA in various organisms will be discussed. Finally, we will report on the role of OGG1 in human disease and the possible use of 8-oxoG DNA N-glycosylases as therapeutic targets.
Assuntos
DNA Glicosilases/metabolismo , DNA-Formamidopirimidina Glicosilase/metabolismo , Proteínas de Escherichia coli/metabolismo , Escherichia coli/fisiologia , Guanina/análogos & derivados , Saccharomyces cerevisiae/fisiologia , Animais , Carcinogênese , DNA Glicosilases/genética , Reparo do DNA , Instabilidade Genômica , Guanina/metabolismo , Humanos , Camundongos , Camundongos Knockout , Estresse OxidativoRESUMO
To characterize proteins that interact with single-stranded/double-stranded (ss/ds) DNA junctions in whole cell free extracts of Saccharomyces cerevisiae, we used [(32)P]-labeled photoreactive partial DNA duplexes containing a 3'-ss/ds-junction (3'-junction) or a 5'-ss/ds-junction (5'-junction). Identification of labeled proteins was achieved by MALDI-TOF mass spectrometry peptide mass fingerprinting and genetic analysis. In wild-type extract, one of the components of the Ddc1-Rad17-Mec3 complex, Ddc1, was found to be preferentially photocrosslinked at a 3'-junction. On the other hand, RPAp70, the large subunit of the replication protein A (RPA), was the predominant crosslinking product at a 5'-junction. Interestingly, ddc1Δ extracts did not display photocrosslinking of RPAp70 at a 5'-junction. The results show that RPAp70 crosslinked to DNA with a 5'-junction is subject to limited proteolysis in ddc1Δ extracts, whereas it is stable in WT, rad17Δ, mec3Δ and mec1Δ extracts. The degradation of the RPAp70-DNA adduct in ddc1Δ extract is strongly reduced in the presence of the proteasome inhibitor MG 132. We also addressed the question of the stability of free RPA, using anti-RPA antibodies. The results show that RPAp70 is also subject to proteolysis without photocrosslinking to DNA upon incubation in ddc1Δ extract. The data point to a novel property of Ddc1, modulating the turnover of DNA binding proteins such as RPAp70 by the proteasome.
Assuntos
Proteínas de Ciclo Celular/metabolismo , DNA de Cadeia Simples/metabolismo , Proteólise , Proteína de Replicação A/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/metabolismo , Proteínas de Ciclo Celular/genética , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Ligação Proteica , Subunidades Proteicas/metabolismo , Proteína de Replicação A/genética , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/genéticaRESUMO
DNA repair mechanisms are critical for maintaining the integrity of genomic DNA, and their loss is associated with cancer predisposition syndromes. Studies in Saccharomyces cerevisiae have played a central role in elucidating the highly conserved mechanisms that promote eukaryotic genome stability. This review will focus on repair mechanisms that involve excision of a single strand from duplex DNA with the intact, complementary strand serving as a template to fill the resulting gap. These mechanisms are of two general types: those that remove damage from DNA and those that repair errors made during DNA synthesis. The major DNA-damage repair pathways are base excision repair and nucleotide excision repair, which, in the most simple terms, are distinguished by the extent of single-strand DNA removed together with the lesion. Mistakes made by DNA polymerases are corrected by the mismatch repair pathway, which also corrects mismatches generated when single strands of non-identical duplexes are exchanged during homologous recombination. In addition to the true repair pathways, the postreplication repair pathway allows lesions or structural aberrations that block replicative DNA polymerases to be tolerated. There are two bypass mechanisms: an error-free mechanism that involves a switch to an undamaged template for synthesis past the lesion and an error-prone mechanism that utilizes specialized translesion synthesis DNA polymerases to directly synthesize DNA across the lesion. A high level of functional redundancy exists among the pathways that deal with lesions, which minimizes the detrimental effects of endogenous and exogenous DNA damage.
Assuntos
Dano ao DNA , Reparo do DNA , DNA Fúngico/genética , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismoRESUMO
Mismatch-repair factors have a prominent role in surveying eukaryotic DNA-replication fidelity and in ensuring correct meiotic recombination. These functions depend on MutL-homolog heterodimers with Mlh1. In humans, MLH1 mutations underlie half of hereditary nonpolyposis colorectal cancers (HNPCCs). Here we report crystal structures of the MutLα (Mlh1-Pms1 heterodimer) C-terminal domain (CTD) from Saccharomyces cerevisiae, alone and in complex with fragments derived from Mlh1 partners. These structures reveal structural rearrangements and additional domains in MutLα as compared to the bacterial MutL counterparts and show that the strictly conserved C terminus of Mlh1 forms part of the Pms1 endonuclease site. The structures of the ternary complexes between MutLα(CTD) and Exo1 or Ntg2 fragments reveal the binding mode of the MIP-box motif shared by several Mlh1 partners. Finally, the structures provide a rationale for the deleterious impact of MLH1 mutations in HNPCCs.
Assuntos
Proteínas Adaptadoras de Transdução de Sinal/química , Enzimas Reparadoras do DNA/química , Endonucleases/química , Proteínas Nucleares/química , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Enzimas Reparadoras do DNA/metabolismo , Endonucleases/metabolismo , Humanos , Modelos Moleculares , Proteína 1 Homóloga a MutL , Proteínas MutL , Proteínas Nucleares/metabolismo , Estrutura Secundária de ProteínaRESUMO
In Saccharomyces cerevisiae, inactivation of base excision repair (BER) AP endonucleases (Apn1p and Apn2p) results in constitutive phosphorylation of Rad53p and delay in cell cycle progression at the G2/M transition. These data led us to investigate genetic interactions between Apn1p, Apn2p and DNA damage checkpoint proteins. The results show that mec1 sml1, rad53 sml1 and rad9 is synthetic lethal with apn1 apn2. In contrast, apn1 apn2 rad17, apn1 apn2 ddc1 and apn1 apn2 rad24 triple mutants are viable, although they exhibit a strong Can(R) spontaneous mutator phenotype. In these strains, high Can(R) mutation rate is dependent upon functional uracil DNA N-glycosylase (Ung1p) and mutation spectra are dominated by AT to CG events. The results point to a role for Rad17-Mec3-Ddc1 (9-1-1) checkpoint clamp in the prevention of mutations caused by abasic (AP) sites linked to incorporation of dUTP into DNA followed by the excision of uracil by Ung1p. The antimutator role of the (9-1-1) clamp can either rely on its essential function in the induction of the DNA damage checkpoint or to another function that specifically impacts DNA repair and/or mutagenesis at AP sites. Here, we show that the abrogation of the DNA damage checkpoint is not sufficient to enhance spontaneous mutagenesis in the apn1 apn2 rad9 sml1 quadruple mutant. Spontaneous mutagenesis was also explored in strains deficient in the two major DNA N-glycosylases/AP-lyases (Ntg1p and Ntg2p). Indeed, apn1 apn2 ntg1 ntg2 exhibits a strong Ung1p-dependent Can(R) mutator phenotype with a spectrum enriched in AT to CG, like apn1 apn2 rad17. However, genetic analysis reveals that ntg1 ntg2 and rad17 are not epistatic for spontaneous mutagenesis in apn1 apn2. We conclude that under normal growth conditions, dUTP incorporation into DNA is a major source of AP sites that cause high genetic instability in the absence of BER factors (Apn1p, Apn2p, Ntg1p and Ntg2p) and Rad17-Mec3-Ddc1 (9-1-1) checkpoint clamp in yeast.
Assuntos
Dano ao DNA , Reparo do DNA/genética , DNA Fúngico/metabolismo , Nucleotídeos de Desoxiuracil/metabolismo , Mutação/genética , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/genética , Pontos de Checagem do Ciclo Celular , Viabilidade Microbiana , Mutagênese , Taxa de Mutação , Saccharomyces cerevisiae/citologia , Saccharomyces cerevisiae/enzimologiaRESUMO
To characterize proteins that interact with base excision/single-strand interruption repair DNA intermediates in cell free extracts of Saccharomyces cerevisiae, we used a combination of photoaffinity labeling with the protein identification by MALDI-TOF-MS peptide mapping. Photoreactive analogue of dCTP, namely exo-N-[4-(4-azido-2,3,5,6,-tetrafluorobenzylidenehydrazinocarbonyl)-butylcarbamoyl]-2'-deoxycytidine-5'-triphosphate, and [(32)P]-labeled DNA duplex containing one nucleotide gap were used to generate nick-containing DNA with a photoreactive dCMP residue at the 3'-margin of the nick. This photoreactive DNA derivative was incubated with the yeast cell extract and after UV irradiation a number of proteins were labeled. Two of the crosslinked proteins were identified as the catalytic subunit of DNA polymerase É and Ddc1 checkpoint protein. Labeling of DNA polymerase É catalytic subunit with the nick-containing DNA repair intermediate indicates that the DNA polymerase is involved in the DNA repair synthesis in yeast, at least at DNA single-strand interruptions. Crosslinking of Ddc1 to DNA nicks took place independently of the other components of checkpoint clamp, Mec3 and Rad17, suggesting that the protein alone is able to recognize DNA single-strand breaks. Indeed, purified GST-tagged Ddc1 protein was efficiently crosslinked to nick-containing DNA. The interaction of Ddc1 with DNA nicks may provide a link between the DNA damage checkpoint and DNA base excision/single-strand breaks repair pathways in yeast. In addition, we found that absence of Ddc1 protein greatly influences the overall pattern of other proteins crosslinked to DNA nick. We suggested that this last effect of Ddc1 is at least partially due to its capacity to prevent proteolytic degradation of the DNA-protein adducts.
Assuntos
Proteínas de Ciclo Celular/química , Quebras de DNA de Cadeia Simples , DNA Polimerase II/química , Reparo do DNA , DNA Fúngico/química , Proteínas de Saccharomyces cerevisiae/química , Saccharomyces cerevisiae/química , Sequência de Aminoácidos , Domínio Catalítico , Dados de Sequência Molecular , Fragmentos de Peptídeos/química , Ligação Proteica , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz , Coloração e RotulagemRESUMO
High transcription is associated with genetic instability, notably increased spontaneous mutation rates, which is a phenomenon termed Transcription-Associated-Mutagenesis (TAM). In this study, we investigated TAM using the chromosomal CAN1 gene under the transcriptional control of two strong and inducible promoters (pGAL1 and pTET) in Saccharomyces cerevisiae. Both pTET- and pGAL1-driven high transcription at the CAN1 gene result in enhanced spontaneous mutation rates. Comparison of both promoters reveals differences in the type of mutagenesis, except for short (-2 and -3 nt) deletions, which depend only on the level of transcription. This mutation type, characteristic of TAM, is sequence dependent, occurring prefentially at di- and trinucleotides repeats, notably at two mutational hotspots encompassing the same 5'-ACATAT-3' sequence. To explore the mechanisms underlying the formation of short deletions in the course of TAM, we have determined Can(R) mutation spectra in yeast mutants affected in DNA metabolism. We identified topoisomerase 1-deficient strains (top1Δ) that specifically abolish the formation of short deletions under high transcription. The rate of the formation of (-2/-3nt) deletions is also reduced in the absence of RAD1 and MUS81 genes, involved in the repair of Top1p-DNA covalent complex. Furthermore ChIP analysis reveals an enrichment of trapped Top1p in the CAN1 ORF under high transcription. We propose a model, in which the repair of trapped Top1p-DNA complexes provokes the formation of short deletion in S. cerevisiae. This study reveals unavoidable conflicts between Top1p and the transcriptional machinery and their potential impact on genome stability.
Assuntos
Sistemas de Transporte de Aminoácidos Básicos/genética , DNA Topoisomerases Tipo I/genética , Deleção de Genes , Regulação Fúngica da Expressão Gênica , Proteínas de Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/genética , DNA/genética , Análise Mutacional de DNA , Modelos Genéticos , Mutagênese , Mutação , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Deleção de Sequência , Transcrição GênicaRESUMO
The Rad2/XPG family nuclease, Exo1, functions in a variety of DNA repair pathways. During meiosis, Exo1 promotes crossover recombination and thereby facilitates chromosome segregation at the first division. Meiotic recombination is initiated by programmed DNA double-strand breaks (DSBs). Nucleolytic resection of DSBs generates long 3' single-strand tails that undergo strand exchange with a homologous chromosome to form joint molecule (JM) intermediates. We show that meiotic DSB resection is dramatically reduced in exo1Δ mutants and test the idea that Exo1-catalyzed resection promotes crossing over by facilitating formation of crossover-specific JMs called double Holliday junctions (dHJs). Contrary to this idea, dHJs form at wild-type levels in exo1Δ mutants, implying that Exo1 has a second function that promotes resolution of dHJs into crossovers. Surprisingly, the dHJ resolution function of Exo1 is independent of its nuclease activities but requires interaction with the putative endonuclease complex, Mlh1-Mlh3. Thus, the DSB resection and procrossover functions of Exo1 during meiosis involve temporally and biochemically distinct activities.
Assuntos
Troca Genética , Quebras de DNA de Cadeia Dupla , Reparo do DNA , Exodesoxirribonucleases/metabolismo , Meiose , Saccharomyces cerevisiae/enzimologia , Saccharomyces cerevisiae/genética , Biocatálise , Exodesoxirribonucleases/genética , Mutação , Saccharomyces cerevisiae/citologiaRESUMO
7,8-Dihydro-8-oxoguanine (8-oxoG) is an abundant and mutagenic DNA lesion. In Saccharomyces cerevisiae, the 8-oxoG DNA N-glycosylase (Ogg1) acts as the primary defense against 8-oxoG. Here, we present evidence for cooperation between Rad18-Rad6-dependent monoubiquitylation of PCNA at K164, the damage-tolerant DNA polymerase eta and the mismatch repair system (MMR) to prevent 8-oxoG-induced mutagenesis. Preventing PCNA modification at lysine 164 (pol30-K164R) results in a dramatic increase in GC to TA mutations due to endogenous 8-oxoG in Ogg1-deficient cells. In contrast, deletion of RAD5 or SIZ1 has little effect implying that the modification of PCNA relevant for preventing 8-oxoG-induced mutagenesis is monoubiquitin as opposed to polyubiquitin or SUMO. We also report that the ubiquitin-binding domain (UBZ) of Pol eta is essential to prevent 8-oxoG-induced mutagenesis but only in conjunction with a functional PCNA-binding domain (PIP). We propose that PCNA is ubiquitylated during the repair synthesis reaction after the MMR-dependent excision of adenine incorporated opposite to 8-oxoG. Monoubiquitylation of PCNA would favor the recruitment of Pol eta thereby allowing error-free incorporation of dCMP opposite to 8-oxoG. This study suggests that Pol eta and the post-replication repair (PRR) machinery can also prevent mutagenesis at DNA lesions that do not stall replication forks.
Assuntos
DNA Polimerase Dirigida por DNA/genética , Guanina/análogos & derivados , Mutagênese , Antígeno Nuclear de Célula em Proliferação/metabolismo , Saccharomyces cerevisiae/genética , Ubiquitinação , Sítios de Ligação , Canavanina/farmacologia , DNA Glicosilases/genética , DNA Glicosilases/metabolismo , Reparo do DNA , DNA Polimerase Dirigida por DNA/química , DNA Polimerase Dirigida por DNA/metabolismo , Deleção de Genes , Guanina/metabolismo , Mutação , Antígeno Nuclear de Célula em Proliferação/química , Estrutura Terciária de Proteína , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/genética , Ubiquitina/metabolismoRESUMO
Mlh1 is an essential factor of mismatch repair (MMR) and meiotic recombination. It interacts through its C-terminal region with MutL homologs and proteins involved in DNA repair and replication. In this study, we identified the site of yeast Mlh1 critical for the interaction with Exo1, Ntg2, and Sgs1 proteins, designated as site S2 by reference to the Mlh1/Pms1 heterodimerization site S1. We show that site S2 is also involved in the interaction between human MLH1 and EXO1 or BLM. Binding at this site involves a common motif on Mlh1 partners that we called the MIP-box for the Mlh1 interacting protein box. Direct and specific interactions between yeast Mlh1 and peptides derived from Exo1, Ntg2, and Sgs1 and between human MLH1 and peptide derived from EXO1 and BLM were measured with K(d) values ranging from 8.1 to 17.4 microM. In Saccharomyces cerevisiae, a mutant of Mlh1 targeted at site S2 (Mlh1-E682A) behaves as a hypomorphic form of Exo1. The site S2 in Mlh1 mediates Exo1 recruitment in order to optimize MMR-dependent mutation avoidance. Given the conservation of Mlh1 and Exo1 interaction, it may readily impact Mlh1-dependent functions such as cancer prevention in higher eukaryotes.
Assuntos
Proteínas Adaptadoras de Transdução de Sinal/química , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Sequência Conservada , Reparo de Erro de Pareamento de DNA , Exodesoxirribonucleases/metabolismo , Proteínas Nucleares/química , Proteínas Nucleares/metabolismo , Proteínas de Saccharomyces cerevisiae/química , Proteínas de Saccharomyces cerevisiae/metabolismo , Motivos de Aminoácidos , Sequência de Aminoácidos , Aminoácidos/metabolismo , Sítios de Ligação , Calorimetria , Dimerização , Humanos , Modelos Moleculares , Dados de Sequência Molecular , Proteína 1 Homóloga a MutL , Mutagênese Sítio-Dirigida , Mutação/genética , Peptídeos/metabolismo , Ligação Proteica , Saccharomyces cerevisiae/enzimologia , Deleção de Sequência , Especificidade da Espécie , Relação Estrutura-Atividade , Técnicas do Sistema de Duplo-HíbridoRESUMO
BACKGROUND: (5R) and (5S) diastereomers of 1-[2-deoxy-beta-D-erythro-pentofuranosyl]-5-hydroxyhydantoin (5-OH-dHyd) and 1-[2-deoxy-beta-D-erythro-pentofuranosyl]-5-hydroxy-5-methylhydantoin (5-OH-5-Me-dHyd) are major oxidation products of 2'-deoxycytidine and thymidine respectively. If not repaired, when present in cellular DNA, these base lesions may be processed by DNA polymerases that induce mutagenic and cell lethality processes. METHODS: Synthetic oligonucleotides that contained a unique 5-hydroxyhydantoin (5-OH-Hyd) or 5-hydroxy-5-methylhydantoin (5-OH-5-Me-Hyd) nucleobase were used as probes for repair studies involving several E. coli, yeast and human purified DNA N-glycosylases. Enzymatic reaction mixtures were analyzed by denaturing polyacrylamide gel electrophoresis after radiolabeling of DNA oligomers or by MALDI-TOF mass spectrometry measurements. RESULTS: In vitro DNA excision experiments carried out with endo III, endo VIII, Fpg, Ntg1 and Ntg2, show that both base lesions are substrates for these DNA N-glycosylases. The yeast and human Ogg1 proteins (yOgg1 and hOgg1 respectively) and E. coli AlkA were unable to cleave the N-glycosidic bond of the 5-OH-Hyd and 5-OH-5-Me-Hyd lesions. Comparison of the kcat/Km ratio reveals that 8-oxo-7,8-dihydroguanine is only a slightly better substrate than 5-OH-Hyd and 5-OH-5-Me-Hyd. The kinetic results obtained with endo III indicate that 5-OH-Hyd and 5-OH-5-Me-Hyd are much better substrates than 5-hydroxycytosine, a well known oxidized pyrimidine substrate for this DNA N-glycosylase. CONCLUSIONS: The present study supports a biological relevance of the base excision repair processes toward the hydantoin lesions, while the removal by the Fpg and endo III proteins are effected at better or comparable rates to that of the removal of 8-oxoGua and 5-OH-Cyt, two established cellular substrates. GENERAL SIGNIFICANCE: The study provides new insights into the substrate specificity of DNA N-glycosylases involved in the base excision repair of oxidized bases, together with complementary information on the biological role of hydantoin type lesions.
Assuntos
DNA Glicosilases/química , Proteínas de Escherichia coli/química , Hidantoínas/química , Pirimidinas/química , Proteínas de Saccharomyces cerevisiae/química , Dano ao DNA , Reparo do DNA , Desoxirribonuclease (Dímero de Pirimidina)/química , Humanos , Cinética , Oligonucleotídeos/química , Oxirredução , Estereoisomerismo , Especificidade por SubstratoRESUMO
Cadmium (Cd(2+)) is a ubiquitous environmental pollutant and human carcinogen. The molecular basis of its toxicity remains unclear. Here, to identify the landscape of genes and cell functions involved in cadmium resistance, we have screened the Saccharomyces cerevisiae deletion collection for mutants sensitive to cadmium exposure. Among the 4866 ORFs tested, we identified 73 genes whose inactivation confers increased sensitivity to Cd(2+). Most were previously unknown to play a role in cadmium tolerance and we observed little correlation between the cadmium sensitivity of a gene deletant and the variation in the transcriptional activity of that gene in response to cadmium. These genes encode proteins involved in various functions: intracellular transport, stress response and gene expression. Analysis of the sensitive phenotype of our "Cd(2+)-sensitive mutant collection" to arsenite, cobalt, mercury and H(2)O(2) revealed 17 genes specifically involved in cadmium-induced response. Among them we found RAD27 and subsequently DNA2 which encode for proteins involved in DNA repair and replication. Analysis of the Cd(2+)-sensitivity of RAD27 (rad27-G67S) and DNA2 (dna2-1) separation of function alleles revealed that their activities necessary for Okazaki fragment processing are essential in conditions of cadmium exposure. Consistently, we observed that wild-type cells exposed to cadmium display an enhanced frequency of forward mutations to canavanine resistance and minisatellite destabilisation. Taken together these results provide a global picture of the genetic requirement for cadmium tolerance in yeast and strongly suggest that DNA replication, through the step of Okazaki fragment processing, is a target of cadmium toxicity.
Assuntos
Adaptação Fisiológica/genética , Cádmio/toxicidade , Replicação do DNA/efeitos dos fármacos , Genes Fúngicos , Saccharomyces cerevisiae/genética , Instabilidade Genômica , Humanos , Saccharomyces cerevisiae/efeitos dos fármacos , Saccharomyces cerevisiae/fisiologia , Sequências de Repetição em TandemRESUMO
In this report we show that human DNA Topoisomerase I (Top1) forms DNA-protein adducts with nicked and gapped DNA structures lacking a conventional Top1 cleavage site. The radioactively labeled crosslinking products were identified by SDS-gel electrophoresis. The chemical structure of the groups at 5' or 3' end of the nick does not have an effect on the formation of these covalent adducts. Therefore, all kinds of nicks, either directly induced by ionizing radiation or reactive oxygen species or indirectly induced in the course of base excision repair (BER) are targets for Top1 that competes with BER proteins and other nick-sensors. Top1-DNA covalent adducts formed in cells exposed to DNA damaging agents can promote genetic instability.
Assuntos
Adutos de DNA/isolamento & purificação , Reparo do DNA , DNA Topoisomerases Tipo I/isolamento & purificação , DNA/química , Reagentes de Ligações Cruzadas/química , Quebras de DNA de Cadeia Simples , HumanosRESUMO
Replication forks stall at DNA lesions or as a result of an unfavorable replicative environment. These fork stalling events have been associated with recombination and gross chromosomal rearrangements. Recombination and fork bypass pathways are the mechanisms accountable for restart of stalled forks. An important lesion bypass mechanism is the highly conserved post-replication repair (PRR) pathway that is composed of error-prone translesion and error-free bypass branches. EXO1 codes for a Rad2p family member nuclease that has been implicated in a multitude of eukaryotic DNA metabolic pathways that include DNA repair, recombination, replication, and telomere integrity. In this report, we show EXO1 functions in the MMS2 error-free branch of the PRR pathway independent of the role of EXO1 in DNA mismatch repair (MMR). Consistent with the idea that EXO1 functions independently in two separate pathways, we defined a domain of Exo1p required for PRR distinct from those required for interaction with MMR proteins. We then generated a point mutant exo1 allele that was defective for the function of Exo1p in MMR due to disrupted interaction with Mlh1p, but still functional for PRR. Lastly, by using a compound exo1 mutant that was defective for interaction with Mlh1p and deficient for nuclease activity, we provide further evidence that Exo1p plays both structural and catalytic roles during MMR.
Assuntos
Reparo de Erro de Pareamento de DNA , Replicação do DNA/fisiologia , Exodesoxirribonucleases/genética , Exodesoxirribonucleases/fisiologia , Mutação , Alelos , Replicação do DNA/efeitos dos fármacos , Exodesoxirribonucleases/metabolismo , Metanossulfonato de Metila/farmacologia , Saccharomyces cerevisiae/enzimologiaRESUMO
Apurinic/apyrimidinic (AP) sites are expected to be one of the most frequent endogenous lesions in DNA. AP sites are potentially lethal and mutagenic. Data shows that the simultaneous inactivation of two AP endonucleases (Apn1 and Apn2) and of the nuclease Rad1-Rad10 causes cell death in Saccharomyces cerevisiae. We suggest that the essential function of Apn1, Apn2, and Rad1-Rad10 is to repair endogenous AP sites and related 3'-blocked single strand breaks. This data led us to conclude that the burden of endogenous AP sites is not compatible with life in absence of DNA repair. This chapter describes two genetic assays to investigate origin, repair, and biological consequences of endogenous AP sites in yeast. The first assay relies on genetic crosses and tetrad analysis and uses the apn1 apn2 rad1 triple mutant. The apn1 apn2 rad1 triple mutant is unviable; however, it can form microcolonies. By means of genetic crosses, apn1 apn2 rad1 x quadruple mutants are generated. The size of the colonies formed by each quadruple mutant is compared to that of the apn1 apn2 rad1 triple mutant. Three classes of genes (x) were identified: (i) genes whose inactivation aggravates the phenotype (reduces microcolony size), such as RAD9, RAD50, RAD51, RAD52, MUS81, and MRE11; (ii) genes whose inactivation alleviates the phenotype, such as UNG1, NTG1, and NTG2; and (iii) genes whose inactivation is neutral, such as MAG1 or OGG1. The second assay uses the apn1 apn2 rad14 triple mutant, which is viable but exhibits a spontaneous mutator phenotype. This mutant was used in a colethal screen. This assay allowed the identification of mutation in DNA repair genes such as RAD1 or RAD50, as well as a mutation in the DUT1 gene coding for the dUTPase, which has impact on the formation of AP sites in DNA. A model that summarizes our present and puzzling data on the origin and repair of endogenous AP sites is also presented.
Assuntos
Dano ao DNA , Reparo do DNA , Saccharomyces cerevisiae/genética , Sequência de Bases , DNA/química , DNA/metabolismo , Enzimas Reparadoras do DNA , DNA Liase (Sítios Apurínicos ou Apirimidínicos)/genética , DNA Liase (Sítios Apurínicos ou Apirimidínicos)/metabolismo , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Endodesoxirribonucleases/genética , Endodesoxirribonucleases/metabolismo , Endonucleases/genética , Endonucleases/metabolismo , Dados de Sequência Molecular , Mutação , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo , Endonucleases Específicas para DNA e RNA de Cadeia SimplesRESUMO
The aim of the present study was to identify proteins that bind nicked DNA intermediates formed in the course of base excision repair (BER) in cell free extracts of Saccharomyces cerevisiae. In mammalian cells, nicks in DNA are targets of proteins such as PARP-1 or XRCC1 that have no homologues in yeast. One of the most promising methodologies to trap proteins that interact with damaged DNA lies in using a photocrosslinking technique with photoactivable dNTP analogues such as exo-N-{2-[N-(4-azido-2,5-difluoro-3-chloropyridine-6-yl)-3-aminopropionyl]-aminoethyl}-2'-deoxycytidine-5'-triphosphate (FAP-dCTP) for enzymatic synthesis of DNA probes with a photoreactive dNMP residue at the 3'-margin of a nick. Using this approach, we identified a major covalent DNA-protein adduct between a nick-containing 34-mer DNA duplex and a protein of a molecular mass of around 100-kDa. Unexpectedly, the formation of the 100-kDa adduct did not require the incorporation of the photoreactive dNMP residue at the 3'-margin of the nick nor exposure to near UV-light. However, the formation of the 100-kDa adduct strictly required a nick or a short gap in the DNA probe. Furthermore, the 100-kDa adduct was not detected in yeast extracts lacking DNA topoisomerase I (Top1). To further establish the nature of crosslinked protein, yeast Top1 was tagged with a Myc-epitope. In this case, the mobility of the Top1-DNA adduct increased by 7- kDa. Therefore, our data speak in favor of Top1 trapping by nicked DNA. In support of this hypothesis, purified yeast Top1 was also crosslinked to nicked DNA structures. Undamaged, uracil- and abasic (AP) site-containing DNAs were unable to trap Top1 under the same assay conditions. Since nicked DNA structures are frequently formed in the course of BER, their covalent linkage to Top1 has the potential to interfere with BER in vivo.
Assuntos
DNA Topoisomerases Tipo I/metabolismo , DNA Fúngico/metabolismo , Saccharomyces cerevisiae/metabolismo , Marcadores de Afinidade , Sequência de Bases , Sistema Livre de Células , Reagentes de Ligações Cruzadas , Dano ao DNA , DNA Polimerase beta/metabolismo , Reparo do DNA , DNA Fúngico/química , DNA Fúngico/genética , Poli(ADP-Ribose) Polimerases/metabolismo , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo , Especificidade por SubstratoRESUMO
We identified a viable allele (dut1-1) of the DUT1 gene that encodes the dUTPase activity in Saccharomyces cerevisiae. The Dut1-1 protein possesses a single amino acid substitution (Gly82Ser) in a conserved motif nearby the active site and exhibits a greatly reduced dUTPase activity. The dut1-1 single mutant exhibits growth delay and cell cycle abnormalities and shows a strong spontaneous mutator phenotype. All phenotypes of the dut1-1 mutant are suppressed by the simultaneous inactivation of the uracil DNA N-glycosylase, Ung1. However, the ung1 dut1-1 double mutant accumulates uracil in its genomic DNA. The viability of the dut1-1 mutant is greatly impaired by the simultaneous inactivation of AP endonucleases. These data strongly suggest that the phenotypes of the dut1-1 mutant result from the incorporation of dUMPs into DNA subsequently converted into AP sites. The analysis of the dut1-1 strain mutation spectrum showed that cytosines are preferentially incorporated in front of AP sites in a Rev3-dependent manner during translesion synthesis. These results point to a critical role of the Dut1 protein in the maintenance of the genetic stability. Therefore, the normal cellular metabolism, and not only its byproducts, is an important source of endogenous DNA damage and genetic instability in eukaryotic cells.
Assuntos
Dano ao DNA , Instabilidade Genômica , Pirofosfatases/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/enzimologia , Saccharomyces cerevisiae/genética , Alelos , Sequência de Aminoácidos , Substituição de Aminoácidos , DNA Fúngico/química , DNA Fúngico/metabolismo , Deleção de Genes , Dados de Sequência Molecular , Mutação , Pirofosfatases/genética , Proteínas de Saccharomyces cerevisiae/genética , Uracila/metabolismoRESUMO
Repair of chemically modified bases in DNA is accomplished through base excision repair (BER). This pathway is initiated by a specific DNA glycosylase that recognizes and excises the altered base to yield an abasic (AP) site. After cleavage of the AP site by APE1, repair proceeds through re-synthesis and ligation steps. In mammalian cells, the XRCC1 protein, essential for the maintenance of genomic stability, is involved in both base excision and single-strand break repair. XRCC1 participates in the first step of BER by interacting with the human DNA glycosylases hOGG1 and NEIL1. To analyze the possibility of a general mechanism involving the interaction of XRCC1 with DNA glycosylases we used XRCC1 to pull-down DNA glycosylases activities from human cell extracts. XRCC1 co-purifies with DNA glycosylase activities capable of excising hypoxanthine and dihydrothymine, in addition to 8-oxoguanine, but not uracil. Biochemical analyses with the purified proteins confirmed the interactions between XRCC1 and MPG, hNTH1 or hNEIL2. Furthermore, XRCC1 stimulates the activities of these enzymes. In vivo localization studies show that after genotoxic treatments these DNA glycosylases can be found associated with XRCC1 foci. Our results support a BER model in which XRCC1 is recruited to the repair of alkylated or oxidized bases by the enzyme recognizing the lesion. XRCC1 would then coordinate the subsequent enzymatic steps and modulate the activities of all the proteins involved.
Assuntos
DNA Glicosilases/metabolismo , Reparo do DNA , Proteínas de Ligação a DNA/metabolismo , Núcleo Celular/química , DNA Glicosilases/análise , Proteínas de Ligação a DNA/análise , Guanina/análogos & derivados , Guanina/metabolismo , Células HeLa , Humanos , Hipoxantinas/metabolismo , Mapeamento de Interação de Proteínas , Proteína 1 Complementadora Cruzada de Reparo de Raio-XRESUMO
To improve the analyses of a form of oxidative DNA damage, 8-hydroxyguanine (8-OH-Gua), we treated isolated DNA with formamidopyrimidine DNA glycosylase (Fpg) and analyzed the released 8-OH-Gua by using a high-performance liquid chromatography system equipped with an electrochemical detector (HPLC-ECD). The human lung carcinoma cells (A549) and human keratinocyte (HaCaT) were irradiated with gamma-rays. After the isolated DNA was treated with the Fpg protein, we analyzed the released 8-OH-Gua by using an HPLC-ECD. With this method, the background level of 8-OH-Gua in DNA from human lung carcinoma cells was determined to be 3.4 residues per 10(7) guanine (Gua). A similar background level of 8-OH-Gua (3.1 residues per 10(7) Gua) was also detected in human keratinocyte DNA with this method. These background 8-OH-Gua levels in cellular DNA are comparable to that obtained previously by an analysis of 8-OH-dGMP after nuclease P1 digestion of cellular DNA (4.3 residues per 10(7) dCMP). A dose-dependent increase of 8-OH-Gua (0.17/10(7) Gua/Gy) was observed after cells were irradiated with gamma-rays. Twenty hours after gamma-irradiation with 60 Gy, 75% of the 8-OH-Gua produced in keratinocyte DNA was repaired. With our new analysis method, it is possible to detect the small changes in the 8-OH-Gua levels in cellular DNA induced by various environmental factors.