RESUMO
In model DNA, A pairs with T, and C with G. However, in vivo, the complementarity of the DNA strands may be disrupted by errors in DNA replication, biochemical modification of bases and recombination. In prokaryotic organisms, mispaired bases are recognized by MutS homologs which, together with MutL homologs, initiate mismatch repair. These same proteins also participate in base excision repair and nucleotide excision repair. In eukaryotes they regulate not just DNA repair but also meiotic recombination, cell-cycle delay and/or apoptosis in response to DNA damage, and hypermutation in immunoglobulin genes. Significantly, the same DNA mismatches that trigger repair in some circumstances trigger non-repair pathways in others. In this review, we argue that mismatch recognition by the MutS proteins is linked to these disparate biological outcomes through regulated interaction of MutL proteins with a wide variety of effector proteins.
Assuntos
Adenosina Trifosfatases/genética , Dano ao DNA , Reparo do DNA , DNA/genética , Proteínas de Escherichia coli/genética , Recombinação Genética , Trifosfato de Adenosina/química , Apoptose , Pareamento Incorreto de Bases , Ciclo Celular , Escherichia coli/genética , Meiose , Modelos Genéticos , Proteínas MutL , Proteína MutS de Ligação de DNA com Erro de Pareamento/genética , MutaçãoRESUMO
Base pair mismatches in DNA arise from errors in DNA replication, recombination, and biochemical modification of bases. Mismatches are inherently transient. They are resolved passively by DNA replication, or actively by enzymatic removal and resynthesis of one of the bases. The first step in removal is recognition of strand discontinuity by one of the MutS proteins. Mismatches arising from errors in DNA replication are repaired in favor of the base on the template strand, but other mismatches trigger base excision or nucleotide excision repair (NER), or non-repair pathways such as hypermutation, cell cycle arrest, or apoptosis. We argue that MutL homologues play a key role in determining biologic outcome by recruiting and/or activating effector proteins in response to lesion recognition by MutS. We suggest that the process is regulated by conformational changes in MutL caused by cycles of ATP binding and hydrolysis, and by physiologic changes which influence effector availability.
Assuntos
Adenosina Trifosfatases/metabolismo , Reparo de Erro de Pareamento de DNA/fisiologia , Proteínas de Escherichia coli/metabolismo , Escherichia coli/metabolismo , Adenosina Trifosfatases/química , Trifosfato de Adenosina/metabolismo , Animais , DNA Glicosilases/metabolismo , DNA Helicases/metabolismo , Reparo de Erro de Pareamento de DNA/genética , Replicação do DNA , DNA Bacteriano/genética , DNA Bacteriano/metabolismo , Endodesoxirribonucleases/metabolismo , Escherichia coli/genética , Proteínas de Escherichia coli/química , Modelos Biológicos , Proteínas MutL , Proteína MutS de Ligação de DNA com Erro de Pareamento/metabolismo , Conformação Proteica , Transdução de Sinais , Hipermutação Somática de ImunoglobulinaRESUMO
In Escherichia coli, T/G mismatches arising from deamination of 5-methylcytosine to thymine are converted to CG base pairs by the very short patch (VSP) repair pathway. DNA Polymerase I removes and resynthesizes the mismatched T starting from a 5'-nick created by the Vsr endonuclease. We used limited trypsinolysis to probe conformational changes in the N-terminal domain of Vsr in response to DNA binding, DNA cleavage and interaction with the polymerase. Our data show that the domain becomes trypsin resistant only under conditions that allow DNA cleavage, while interaction with the polymerase restores trypsin sensitivity. We suggest that the domain changes its conformation as a result of DNA nicking, and that DNA Pol I releases Vsr from the nick by reversing that conformational change.
Assuntos
Clivagem do DNA , Endodesoxirribonucleases/química , Endodesoxirribonucleases/metabolismo , DNA Polimerase I/química , DNA Polimerase I/metabolismo , Conformação Proteica , Estrutura Terciária de ProteínaRESUMO
The activities of the Vsr and MutH endonucleases of Escherichia coli are stimulated by MutL. The interaction of MutL with each enzyme is enhanced in vivo by 2-aminopurine treatment and by inactivation of the mutY gene. We hypothesize that MutL recruits the endonucleases to sites of DNA damage.
Assuntos
Adenosina Trifosfatases/metabolismo , Dano ao DNA , Enzimas Reparadoras do DNA/metabolismo , Proteínas de Ligação a DNA/metabolismo , Endodesoxirribonucleases/metabolismo , Proteínas de Escherichia coli/metabolismo , Escherichia coli/enzimologia , Escherichia coli/genética , Adenosina Trifosfatases/genética , Reparo de Erro de Pareamento de DNA , Enzimas Reparadoras do DNA/genética , Proteínas de Ligação a DNA/genética , Endodesoxirribonucleases/genética , Proteínas de Escherichia coli/genética , Proteínas MutL , Ligação ProteicaRESUMO
5-Azacytidine induces CG-to-GC transversion mutations in Escherichia coli. The results presented in this paper provide evidence that repair of the drug-induced lesions that produce these mutations involves components of both the mismatch repair and nucleotide excision repair systems. Strains deficient in mutL, mutS, uvrA, uvrB or uvrC all showed an increase in mutation in response to 5-azacytidine. Using a bacterial two-hybrid assay, we showed that UvrB interacts with MutL and MutS in a drug-dependent manner, while UvrC interacts with MutL independent of drug. We suggest that 5-azacytidine-induced mismatches recruit MutS and MutL, but are poorly processed by mismatch repair. Instead, the stalled MutS-MutL complex recruits the Uvr proteins to complete repair.
Assuntos
Azacitidina/administração & dosagem , Pareamento Incorreto de Bases/efeitos dos fármacos , Reparo de Erro de Pareamento de DNA/fisiologia , Reparo do DNA/fisiologia , DNA Bacteriano/efeitos dos fármacos , DNA Bacteriano/metabolismo , Escherichia coli/metabolismo , Adenosina Trifosfatases/deficiência , Adenosina Trifosfatases/efeitos dos fármacos , DNA Helicases/deficiência , DNA Helicases/efeitos dos fármacos , Enzimas Reparadoras do DNA/deficiência , Enzimas Reparadoras do DNA/efeitos dos fármacos , DNA Bacteriano/genética , Proteínas de Ligação a DNA/deficiência , Proteínas de Ligação a DNA/efeitos dos fármacos , DNA-Citosina Metilases/metabolismo , Relação Dose-Resposta a Droga , Endodesoxirribonucleases/deficiência , Endodesoxirribonucleases/efeitos dos fármacos , Inibidores Enzimáticos/administração & dosagem , Escherichia coli/efeitos dos fármacos , Escherichia coli/genética , Proteínas de Escherichia coli/efeitos dos fármacos , Mutação da Fase de Leitura/efeitos dos fármacos , Proteínas MutL , Proteína MutS de Ligação de DNA com Erro de Pareamento/deficiência , Proteína MutS de Ligação de DNA com Erro de Pareamento/efeitos dos fármacos , Técnicas do Sistema de Duplo-HíbridoRESUMO
DNA polymerase lambda (pol lambda) is a member of the X family of DNA polymerases that has been implicated in both base excision repair and non-homologous end joining through in vitro studies. However, to date, no phenotype has been associated with cells deficient in this DNA polymerase. Here we show that pol lambda null mouse fibroblasts are hypersensitive to oxidative DNA damaging agents, suggesting a role of pol lambda in protection of cells against the cytotoxic effects of oxidized DNA. Additionally, pol lambda co-immunoprecipitates with an oxidized base DNA glycosylase, single-strand-selective monofunctional uracil-DNA glycosylase (SMUG1), and localizes to oxidative DNA lesions in situ. From these data, we conclude that pol lambda protects cells against oxidative stress and suggest that it participates in oxidative DNA damage base excision repair.
Assuntos
Dano ao DNA/fisiologia , DNA Polimerase beta/fisiologia , Reparo do DNA/fisiologia , Fibroblastos/fisiologia , Animais , Linhagem Celular , Dano ao DNA/genética , DNA Glicosilases/metabolismo , DNA Polimerase beta/deficiência , DNA Polimerase beta/genética , Reparo do DNA/genética , Células HeLa , Humanos , Camundongos , Oxidantes/química , Oxirredução , Pentoxil (Uracila)/análogos & derivados , Pentoxil (Uracila)/farmacologia , Uracila-DNA GlicosidaseRESUMO
DNA polymerase beta (pol beta) is the major DNA polymerase involved in the base excision repair (BER) pathway in mammalian cells and, as a consequence, BER is severely compromised in cells lacking pol beta. Pol beta null (-/-) mouse embryos are not viable and pol beta null cells are hypersensitive to alkylating agents. Using RNA interference (RNAi) technology in mouse cells, we have reduced the pol beta protein and mRNA to undetectable levels. Pol beta knockdown cell lines display a pattern of hypersensitivity to DNA damaging agents similar to that observed in pol beta null cells. Generation of pol beta knock down cells makes it possible to combine the pol beta null phenotype with deficiencies in other DNA repair proteins, thereby helping to elucidate the role of pol beta and its interactions with other proteins in mammalian cells.
Assuntos
Dano ao DNA , DNA Polimerase beta/deficiência , DNA Polimerase beta/genética , Reparo do DNA , Interferência de RNA , Animais , Sequência de Bases , Bleomicina/toxicidade , Linhagem Celular , Cisplatino/toxicidade , DNA Polimerase beta/metabolismo , DNA Complementar/genética , Peróxido de Hidrogênio/toxicidade , Metanossulfonato de Metila/toxicidade , Azul de Metileno/toxicidade , Camundongos , Fenótipo , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Raios Ultravioleta/efeitos adversosRESUMO
Nucleoside diphosphate kinase from the haloalkaliphilic archaeon Natrialba magadii (Nm NDPK) is a homooligomeric hexamer with a monomer molecular weight of 23 kDa. Its main function is to exchange gamma-phosphates between nucleoside triphosphates and diphosphates. Previously it was shown that Nm NDPK is active over a wide range of NaCl concentrations, which is not typical of extremely halophilic proteins. In this paper more detailed investigations of kinase function and stability were carried out using circular dichroism, differential scanning calorimetry, size-exclusion chromatography, and biochemical methods. A possible mechanism for stabilization of halophilic proteins that allows them to function in a wide range of NaCl concentrations is proposed.
