RESUMO
The Msh2-Msh3 mismatch repair (MMR) complex in Saccharomyces cerevisiae recognizes and directs repair of insertion/deletion loops (IDLs) up to â¼17 nucleotides. Msh2-Msh3 also recognizes and binds distinct looped and branched DNA structures with varying affinities, thereby contributing to genome stability outside post-replicative MMR through homologous recombination, double-strand break repair (DSBR) and the DNA damage response. In contrast, Msh2-Msh3 promotes genome instability through trinucleotide repeat (TNR) expansions, presumably by binding structures that form from single-stranded (ss) TNR sequences. We previously demonstrated that Msh2-Msh3 binding to 5' ssDNA flap structures interfered with Rad27 (Fen1 in humans)-mediated Okazaki fragment maturation (OFM) in vitro. Here we demonstrate that elevated Msh2-Msh3 levels interfere with DNA replication and base excision repair in vivo. Elevated Msh2-Msh3 also induced a cell cycle arrest that was dependent on RAD9 and ELG1 and led to PCNA modification. These phenotypes also required Msh2-Msh3 ATPase activity and downstream MMR proteins, indicating an active mechanism that is not simply a result of Msh2-Msh3 DNA-binding activity. This study provides new mechanistic details regarding how excess Msh2-Msh3 can disrupt DNA replication and repair and highlights the role of Msh2-Msh3 protein abundance in Msh2-Msh3-mediated genomic instability.
Assuntos
Instabilidade Genômica , Proteínas de Saccharomyces cerevisiae , Humanos , DNA/genética , DNA/metabolismo , Reparo de Erro de Pareamento de DNA , Reparo do DNA , Proteínas de Ligação a DNA/metabolismo , Proteína 2 Homóloga a MutS/genética , Proteína 2 Homóloga a MutS/metabolismo , Proteína 3 Homóloga a MutS/genética , Proteína 3 Homóloga a MutS/metabolismo , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismoRESUMO
Determining mutation signatures is standard for understanding the etiology of human tumors and informing cancer treatment. Multiple determinants of DNA replication fidelity prevent mutagenesis that leads to carcinogenesis, including the regulation of free deoxyribonucleoside triphosphate pools by ribonucleotide reductase and repair of replication errors by the mismatch repair system. We identified genetic interactions between rnr1 alleles that skew and/or elevate deoxyribonucleoside triphosphate levels and mismatch repair gene deletions. These defects indicate that the rnr1 alleles lead to increased mutation loads that are normally acted upon by mismatch repair. We then utilized a targeted deep-sequencing approach to determine mutational profiles associated with mismatch repair pathway defects. By combining rnr1 and msh mutations to alter and/or increase deoxyribonucleoside triphosphate levels and alter the mutational load, we uncovered previously unreported specificities of Msh2-Msh3 and Msh2-Msh6. Msh2-Msh3 is uniquely able to direct the repair of G/C single-base deletions in GC runs, while Msh2-Msh6 specifically directs the repair of substitutions that occur at G/C dinucleotides. We also identified broader sequence contexts that influence variant profiles in different genetic backgrounds. Finally, we observed that the mutation profiles in double mutants were not necessarily an additive relationship of mutation profiles in single mutants. Our results have implications for interpreting mutation signatures from human tumors, particularly when mismatch repair is defective.
Assuntos
Ribonucleotídeo Redutases , Proteínas de Saccharomyces cerevisiae , Humanos , Desoxirribonucleosídeos , Reparo de Erro de Pareamento de DNA , Reparo do DNA , Proteínas de Ligação a DNA/metabolismo , Mutação , Proteína 2 Homóloga a MutS/genética , Proteína 2 Homóloga a MutS/metabolismo , Proteínas MutS/genética , Proteínas MutS/metabolismo , Ribonucleotídeo Redutases/genética , Ribonucleotídeo Redutases/metabolismo , Proteínas de Saccharomyces cerevisiae/genética , Especificidade por SubstratoRESUMO
Trinucleotide repeat (TNR) tracts are inherently unstable during DNA replication, leading to repeat expansions and/or contractions. Expanded tracts are the cause of over 40 neurodegenerative and neuromuscular diseases. In this chapter, we focus on the (CAG)n and (CTG)n repeat sequences that, when expanded, lead to Huntington's disease (HD) and myotonic dystrophy type 1 (DM1), respectively, as well as a number of other neurodegenerative diseases. TNR tracts in most individuals are relatively small and stable in terms of length. However, TNR tracts become increasingly prone to expansion as tract length increases, eventually leading to very long tracts that disrupt coding (e.g. HD) or noncoding (e.g., DM1) regions of the genome. It is important to understand the early stages in TNR expansions, that is, the transition from small, stable lengths to susceptible threshold lengths. We describe PCR-based in vivo assays, using the model system Saccharomyces cerevisiae, to determine and characterize the dynamic behavior of TNR tracts in the stable and threshold ranges. We also describe a simple in vitro system to assess tract dynamics during 5' single-stranded DNA (ssDNA) flap processing and to assess the role of different DNA metabolism proteins in these dynamics. These assays can ultimately be used to determine factors that influence the early stages of TNR tract expansion.
Assuntos
DNA de Cadeia Simples/genética , Proteínas de Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/genética , Repetições de Trinucleotídeos , Replicação do DNA , Instabilidade Genômica , Humanos , Instabilidade de Microssatélites , Reação em Cadeia da Polimerase , Expansão das Repetições de TrinucleotídeosRESUMO
Regulation of dNTP pools in an intracellular environment is not only vital for DNA replication but also plays a major role in maintaining genomic stability. Ribonucleotide reductase (RNR) catalyzes the rate-limiting step in dNTP synthesis and altered regulation of RNR leads to imbalanced dNTP pools. Increased dNTP levels are mutagenic and have the potential to interfere with pathways that are involved in DNA replication, repair and DNA damage control. However, the mechanisms through which altered dNTP pools affect these pathways are poorly understood. Nonetheless, altered dNTP pools have been identified in a number of cellular contexts, including cancer. In order to interpret and analyze the effects of altered dNTP pools, we need quantitative information about dNTP pools in different genetic and environmental contexts in vivo. Here we describe a high-throughput fluorescence-based assay that uses a qPCR-based approach to quantify dNTP levels for use with Saccharomyces cerevisiae extracts.
Assuntos
Desoxirribonucleotídeos/isolamento & purificação , Ensaios de Triagem em Larga Escala/métodos , Saccharomyces cerevisiae/genética , Reparo do DNA , Desoxirribonucleotídeos/biossíntese , Fluorescência , Mutagênese , Reação em Cadeia da Polimerase em Tempo Real/métodos , Ribonucleotídeo Redutases/metabolismoRESUMO
Double strand DNA break repair (DSBR) comprises multiple pathways. A subset of DSBR pathways, including single strand annealing, involve intermediates with 3' non-homologous tails that must be removed to complete repair. In Saccharomyces cerevisiae, Rad1-Rad10 is the structure-specific endonuclease that cleaves the tails in 3' non-homologous tail removal (3' NHTR). Rad1-Rad10 is also an essential component of the nucleotide excision repair (NER) pathway. In both cases, Rad1-Rad10 requires protein partners for recruitment to the relevant DNA intermediate. Msh2-Msh3 and Saw1 recruit Rad1-Rad10 in 3' NHTR; Rad14 recruits Rad1-Rad10 in NER. We created two rad1 separation-of-function alleles, rad1R203A,K205A and rad1R218A; both are defective in 3' NHTR but functional in NER. In vitro, rad1R203A,K205A was impaired at multiple steps in 3' NHTR. The rad1R218A in vivo phenotype resembles that of msh2- or msh3-deleted cells; recruitment of rad1R218A-Rad10 to recombination intermediates is defective. Interactions among rad1R218A-Rad10 and Msh2-Msh3 and Saw1 are altered and rad1R218A-Rad10 interactions with RPA are compromised. We propose a model in which Rad1-Rad10 is recruited and positioned at the recombination intermediate through interactions, between Saw1 and DNA, Rad1-Rad10 and Msh2-Msh3, Saw1 and Msh2-Msh3 and Rad1-Rad10 and RPA. When any of these interactions is altered, 3' NHTR is impaired.
Assuntos
Enzimas Reparadoras do DNA/metabolismo , Proteínas de Ligação a DNA/metabolismo , Endonucleases/metabolismo , Proteína de Replicação A/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/genética , Endonucleases Específicas para DNA e RNA de Cadeia Simples/metabolismo , Quebras de DNA de Cadeia Dupla , Reparo do DNA por Junção de Extremidades , Enzimas Reparadoras do DNA/genética , Proteínas de Ligação a DNA/genética , Endonucleases/genética , Proteína 2 Homóloga a MutS/metabolismo , Proteína 3 Homóloga a MutS/genética , Proteína 3 Homóloga a MutS/metabolismo , Mutação , Mapeamento de Interação de Proteínas , Proteína de Replicação A/genética , Saccharomyces cerevisiae/efeitos da radiação , Proteínas de Saccharomyces cerevisiae/genética , Endonucleases Específicas para DNA e RNA de Cadeia Simples/genética , Raios UltravioletaRESUMO
Trinucleotide repeat (TNR) tracts are inherently unstable during DNA replication, leading to repeat expansions and/or contractions. Expanded tracts are the cause of over 40 neurodegenerative and neuromuscular diseases. In this chapter, we focus on the (CNG)n repeat sequences that, when expanded, lead to Huntington's disease (HD), myotonic dystrophy type 1 (DM1), and a number of other neurodegenerative diseases. We describe a series of in vivo assays, using the model system Saccharomyces cerevisiae, to determine and characterize the dynamic behavior of TNR tracts that are in the early stages of expansion, i.e., the so-called threshold range. Through a series of time courses and PCR-based assays, dynamic changes in tract length can be observed as a function of time. These assays can ultimately be used to determine how genetic factors influence the process of tract expansion in these early stages.
Assuntos
Saccharomyces cerevisiae/genética , Repetições de Trinucleotídeos , Replicação do DNA , DNA Fúngico/isolamento & purificação , Expressão Gênica , Genes Reporter , Genoma Fúngico , Instabilidade Genômica , Instabilidade de Microssatélites , Taxa de Mutação , Expansão das Repetições de TrinucleotídeosRESUMO
Mlh1-Mlh3 is an endonuclease hypothesized to act in meiosis to resolve double Holliday junctions into crossovers. It also plays a minor role in eukaryotic DNA mismatch repair (MMR). To understand how Mlh1-Mlh3 functions in both meiosis and MMR, we analyzed in baker's yeast 60 new mlh3 alleles. Five alleles specifically disrupted MMR, whereas one (mlh3-32) specifically disrupted meiotic crossing over. Mlh1-mlh3 representatives for each class were purified and characterized. Both Mlh1-mlh3-32 (MMR+, crossover-) and Mlh1-mlh3-45 (MMR-, crossover+) displayed wild-type endonuclease activities in vitro. Msh2-Msh3, an MSH complex that acts with Mlh1-Mlh3 in MMR, stimulated the endonuclease activity of Mlh1-mlh3-32 but not Mlh1-mlh3-45, suggesting that Mlh1-mlh3-45 is defective in MSH interactions. Whole genome recombination maps were constructed for wild-type and MMR+ crossover-, MMR- crossover+, endonuclease defective and null mlh3 mutants in an S288c/YJM789 hybrid background. Compared to wild-type, all of the mlh3 mutants showed increases in the number of noncrossover events, consistent with recombination intermediates being resolved through alternative recombination pathways. Our observations provide a structure-function map for Mlh3 that reveals the importance of protein-protein interactions in regulating Mlh1-Mlh3's enzymatic activity. They also illustrate how defective meiotic components can alter the fate of meiotic recombination intermediates, providing new insights for how meiotic recombination pathways are regulated.
Assuntos
Recombinação Homóloga/genética , Proteína 1 Homóloga a MutL/genética , Proteínas MutL/genética , Mapas de Interação de Proteínas/genética , Proteínas de Saccharomyces cerevisiae/genética , Alelos , Troca Genética , Reparo de Erro de Pareamento de DNA/genética , Genoma Fúngico , Meiose/genética , Saccharomyces cerevisiae/genética , Relação Estrutura-AtividadeRESUMO
Crossing over between homologs is initiated in meiotic prophase by the formation of DNA double-strand breaks that occur throughout the genome. In the major interference-responsive crossover pathway in baker's yeast, these breaks are resected to form 3' single-strand tails that participate in a homology search, ultimately forming double Holliday junctions (dHJs) that primarily include both homologs. These dHJs are resolved by endonuclease activity to form exclusively crossovers, which are critical for proper homolog segregation in Meiosis I. Recent genetic, biochemical, and molecular studies in yeast are consistent with the hypothesis of Mlh1-Mlh3 DNA mismatch repair complex acting as the major endonuclease activity that resolves dHJs into crossovers. However, the mechanism by which the Mlh1-Mlh3 endonuclease is activated is unknown. Here, we provide evidence that Mlh1-Mlh3 does not behave like a structure-specific endonuclease but forms polymers required to generate nicks in DNA. This conclusion is supported by DNA binding studies performed with different-sized substrates that contain or lack polymerization barriers and endonuclease assays performed with varying ratios of endonuclease-deficient and endonuclease-proficient Mlh1-Mlh3. In addition, Mlh1-Mlh3 can generate religatable double-strand breaks and form an active nucleoprotein complex that can nick DNA substrates in trans. Together these observations argue that Mlh1-Mlh3 may not act like a canonical, RuvC-like Holliday junction resolvase and support a novel model in which Mlh1-Mlh3 is loaded onto DNA to form an activated polymer that cleaves DNA.
Assuntos
DNA Cruciforme/metabolismo , Endonuclease PMS2 de Reparo de Erro de Pareamento/metabolismo , Proteína 1 Homóloga a MutL/metabolismo , Proteínas MutL/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Substituição de Aminoácidos , Quebras de DNA de Cadeia Dupla , DNA Circular/química , DNA Circular/metabolismo , DNA Cruciforme/química , Proteínas de Ligação a DNA/química , Proteínas de Ligação a DNA/genética , Ativação Enzimática , Humanos , Hidrólise , Endonuclease PMS2 de Reparo de Erro de Pareamento/química , Endonuclease PMS2 de Reparo de Erro de Pareamento/genética , Peso Molecular , Proteína 1 Homóloga a MutL/química , Proteína 1 Homóloga a MutL/genética , Proteínas MutL/química , Proteínas MutL/genética , Proteína 2 Homóloga a MutS/química , Proteína 2 Homóloga a MutS/genética , Proteína 3 Homóloga a MutS , Mutação , Antígeno Nuclear de Célula em Proliferação/genética , Antígeno Nuclear de Célula em Proliferação/metabolismo , Multimerização Proteica , Proteínas Recombinantes de Fusão/química , Proteínas Recombinantes de Fusão/metabolismo , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismo , Proteína de Replicação C/genética , Proteína de Replicação C/metabolismo , Proteínas de Saccharomyces cerevisiae/química , Proteínas de Saccharomyces cerevisiae/genética , Especificidade por SubstratoRESUMO
DNA-binding proteins search for specific targets via facilitated diffusion along a crowded genome. However, little is known about how crowded DNA modulates facilitated diffusion and target recognition. Here we use DNA curtains and single-molecule fluorescence imaging to investigate how Msh2-Msh3, a eukaryotic mismatch repair complex, navigates on crowded DNA. Msh2-Msh3 hops over nucleosomes and other protein roadblocks, but maintains sufficient contact with DNA to recognize a single lesion. In contrast, Msh2-Msh6 slides without hopping and is largely blocked by protein roadblocks. Remarkably, the Msh3-specific mispair-binding domain (MBD) licences a chimeric Msh2-Msh6(3MBD) to bypass nucleosomes. Our studies contrast how Msh2-Msh3 and Msh2-Msh6 navigate on a crowded genome and suggest how Msh2-Msh3 locates DNA lesions outside of replication-coupled repair. These results also provide insights into how DNA repair factors search for DNA lesions in the context of chromatin.
Assuntos
Dano ao DNA , Reparo de Erro de Pareamento de DNA , DNA Fúngico/metabolismo , Proteínas de Ligação a DNA/metabolismo , Proteína 2 Homóloga a MutS/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Cromatina/metabolismo , Reparo do DNA , Histonas/metabolismo , Microscopia de Fluorescência , Proteína 3 Homóloga a MutS , Nucleossomos/metabolismo , Pontos Quânticos , Saccharomyces cerevisiaeRESUMO
In Saccharomyces cerevisiae, Msh2-Msh3-mediated mismatch repair (MMR) recognizes and targets insertion/deletion loops for repair. Msh2-Msh3 is also required for 3' non-homologous tail removal (3'NHTR) in double-strand break repair. In both pathways, Msh2-Msh3 binds double-strand/single-strand junctions and initiates repair in an ATP-dependent manner. However, we recently demonstrated that the two pathways have distinct requirements with respect to Msh2-Msh3 activities. We identified a set of aromatic residues in the nucleotide binding pocket (FLY motif) of Msh3 that, when mutated, disrupted MMR, but left 3'NHTR largely intact. One of these mutations, msh3Y942A, was predicted to disrupt the nucleotide sandwich and allow altered positioning of ATP within the pocket. To develop a mechanistic understanding of the differential requirements for ATP binding and/or hydrolysis in the two pathways, we characterized Msh2-Msh3 and Msh2-msh3Y942A ATP binding and hydrolysis activities in the presence of MMR and 3'NHTR DNA substrates. We observed distinct, substrate-dependent ATP hydrolysis and nucleotide turnover by Msh2-Msh3, indicating that the MMR and 3'NHTR DNA substrates differentially modify the ATP binding/hydrolysis activities of Msh2-Msh3. Msh2-msh3Y942A retained the ability to bind DNA and ATP but exhibited altered ATP hydrolysis and nucleotide turnover. We propose that both ATP and structure-specific repair substrates cooperate to direct Msh2-Msh3-mediated repair and suggest an explanation for the msh3Y942A separation-of-function phenotype.
Assuntos
Trifosfato de Adenosina/metabolismo , Reparo de Erro de Pareamento de DNA , DNA Fúngico/química , DNA Fúngico/metabolismo , Proteínas de Ligação a DNA/metabolismo , Proteína 2 Homóloga a MutS/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/metabolismo , Domínio Catalítico , Quebras de DNA de Cadeia Dupla , Proteínas de Ligação a DNA/química , Proteínas de Ligação a DNA/genética , Hidrólise , Cinética , Proteína 2 Homóloga a MutS/química , Proteína 2 Homóloga a MutS/genética , Proteína 3 Homóloga a MutS , Mutação , Fenótipo , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/química , Proteínas de Saccharomyces cerevisiae/genética , Especificidade por SubstratoRESUMO
In Saccharomyces cerevisiae, repair of insertion/deletion loops is carried out by Msh2-Msh3-mediated mismatch repair (MMR). Msh2-Msh3 is also required for 3' non-homologous tail removal (3' NHTR) in double-strand break repair. In both pathways, Msh2-Msh3 binds double-strand/single-strand junctions and initiates repair in an ATP-dependent manner. However, the kinetics of the two processes appear different; MMR is likely rapid in order to coordinate with the replication fork, whereas 3' NHTR has been shown to be a slower process. To understand the molecular requirements in both repair pathways, we performed an in vivo analysis of well-conserved residues in Msh3 that are hypothesized to be required for MMR and/or 3' NHTR. These residues are predicted to be involved in either communication between the DNA-binding and ATPase domains within the complex or nucleotide binding and/or exchange within Msh2-Msh3. We identified a set of aromatic residues within the FLY motif of the predicted Msh3 nucleotide binding pocket that are essential for Msh2-Msh3-mediated MMR but are largely dispensable for 3' NHTR. In contrast, mutations in other regions gave similar phenotypes in both assays. Based on these results, we suggest that the two pathways have distinct requirements with respect to the position of the bound ATP within Msh3. We propose that the differences are related, at least in part, to the kinetics of each pathway. Proper binding and positioning of ATP is required to induce rapid conformational changes at the replication fork, but is less important when more time is available for repair, as in 3' NHTR.
Assuntos
Trifosfato de Adenosina/metabolismo , Reparo do DNA , Proteínas de Ligação a DNA/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/enzimologia , Saccharomyces cerevisiae/fisiologia , Sequência de Aminoácidos , Análise Mutacional de DNA , Proteínas de Ligação a DNA/química , Proteínas de Ligação a DNA/genética , Cinética , Modelos Biológicos , Modelos Moleculares , Dados de Sequência Molecular , Proteína 3 Homóloga a MutS , Proteínas Mutantes/química , Proteínas Mutantes/genética , Proteínas Mutantes/metabolismo , Ligação Proteica , Saccharomyces cerevisiae/química , Proteínas de Saccharomyces cerevisiae/química , Proteínas de Saccharomyces cerevisiae/genéticaRESUMO
Trinucleotide repeat (TNR) expansions are the underlying cause of more than 40 neurodegenerative and neuromuscular diseases, including myotonic dystrophy and Huntington's disease. Although genetic evidence points to errors in DNA replication and/or repair as the cause of these diseases, clear molecular mechanisms have not been described. Here, we focused on the role of the mismatch repair complex Msh2-Msh3 in promoting TNR expansions. We demonstrate that Msh2-Msh3 promotes CTG and CAG repeat expansions in vivo in Saccharomyces cerevisiae. Furthermore, we provide biochemical evidence that Msh2-Msh3 directly interferes with normal Okazaki fragment processing by flap endonuclease1 (Rad27) and DNA ligase I (Cdc9) in the presence of TNR sequences, thereby producing small, incremental expansion events. We believe that this is the first mechanistic evidence showing the interplay of replication and repair proteins in the expansion of sequences during lagging-strand DNA replication.
Assuntos
Reparo do DNA , Proteínas de Ligação a DNA/metabolismo , DNA/metabolismo , Proteína 2 Homóloga a MutS/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/metabolismo , Expansão das Repetições de Trinucleotídeos , Adenosina Trifosfatases/genética , Adenosina Trifosfatases/metabolismo , DNA/genética , DNA Ligase Dependente de ATP , DNA Ligases/genética , DNA Ligases/metabolismo , Enzimas Reparadoras do DNA/genética , Enzimas Reparadoras do DNA/metabolismo , Proteínas de Ligação a DNA/genética , Humanos , Doença de Huntington/genética , Doença de Huntington/metabolismo , Proteína 2 Homóloga a MutS/genética , Proteína 3 Homóloga a MutS , Distrofia Miotônica/genética , Distrofia Miotônica/metabolismo , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/genéticaRESUMO
DNA mismatch repair (MMR) is a highly conserved mutation avoidance mechanism that corrects DNA polymerase misincorporation errors. In initial steps in MMR, Msh2-Msh6 binds mispairs and small insertion/deletion loops, and Msh2-Msh3 binds larger insertion/deletion loops. The msh2Δ1 mutation, which deletes the conserved DNA-binding domain I of Msh2, does not dramatically affect Msh2-Msh6-dependent repair. In contrast, msh2Δ1 mutants show strong defects in Msh2-Msh3 functions. Interestingly, several mutations identified in patients with hereditary non-polyposis colorectal cancer map to domain I of Msh2; none have been found in MSH3. To understand the role of Msh2 domain I in MMR, we examined the consequences of combining the msh2Δ1 mutation with mutations in two distinct regions of MSH6 and those that increase cellular mutational load (pol3-01 and rad27). These experiments reveal msh2Δ1-specific phenotypes in Msh2-Msh6 repair, with significant effects on mutation rates. In vitro assays demonstrate that msh2Δ1-Msh6 DNA binding is less specific for DNA mismatches and produces an altered footprint on a mismatch DNA substrate. Together, these results provide evidence that, in vivo, multiple factors insulate MMR from defects in domain I of Msh2 and provide insights into how mutations in Msh2 domain I may cause hereditary non-polyposis colorectal cancer.
Assuntos
Reparo de Erro de Pareamento de DNA , Proteínas de Ligação a DNA/metabolismo , Proteína 2 Homóloga a MutS/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Sequência de Bases , Western Blotting , Pegada de DNA , DNA Fúngico/genética , Proteínas de Ligação a DNA/genética , Desoxirribonuclease I/metabolismo , Ensaio de Desvio de Mobilidade Eletroforética , Dados de Sequência Molecular , Proteína 2 Homóloga a MutS/genética , Mutação , Estrutura Terciária de Proteína , Proteínas de Saccharomyces cerevisiae/genética , Homologia de Sequência do Ácido NucleicoRESUMO
Ribonucleotide reductase (RNR) catalyzes the rate-limiting step in nucleotide biosynthesis and plays a central role in genome maintenance. Although a number of regulatory mechanisms govern RNR activity, the physiologic effect of RNR deregulation had not previously been examined in an animal model. We show here that overexpression of the small RNR subunit potently and selectively induces lung neoplasms in transgenic mice and is mutagenic in cultured cells. Combining RNR deregulation with defects in DNA mismatch repair, the cellular mutation correction system, synergistically increased RNR-induced mutagenesis and carcinogenesis. Moreover, the proto-oncogene K-ras was identified as a frequent mutational target in RNR-induced lung neoplasms. Together, these results show that RNR deregulation promotes lung carcinogenesis through a mutagenic mechanism and establish a new oncogenic activity for a key regulator of nucleotide metabolism. Importantly, RNR-induced lung neoplasms histopathologically resemble human papillary adenocarcinomas and arise stochastically via a mutagenic mechanism, making RNR transgenic mice a valuable model for lung cancer.
Assuntos
Regulação Enzimológica da Expressão Gênica , Regulação Neoplásica da Expressão Gênica , Neoplasias Pulmonares/genética , Ribonucleotídeo Redutases/genética , Células 3T3 , Animais , Proteínas de Ligação a DNA/deficiência , Proteínas de Ligação a DNA/genética , Modelos Animais de Doenças , Éxons , Genes ras , Neoplasias Pulmonares/enzimologia , Camundongos , Camundongos Knockout , Camundongos Transgênicos , Mutação , Proto-Oncogene MasRESUMO
The ability of proteins to locate specific sites or structures among a vast excess of nonspecific DNA is a fundamental theme in biology. Yet the basic principles that govern these mechanisms remain poorly understood. For example, mismatch repair proteins must scan millions of base pairs to find rare biosynthetic errors, and they then must probe the surrounding region to identify the strand discrimination signals necessary to distinguish the parental and daughter strands. To determine how these proteins might function we used single-molecule optical microscopy to answer the following question: how does the mismatch repair complex Msh2-Msh6 interrogate undamaged DNA? Here we show that Msh2-Msh6 slides along DNA via one-dimensional diffusion. These findings indicate that interactions between Msh2-Msh6 and DNA are dominated by lateral movement of the protein along the helical axis and have implications for how MutS family members travel along DNA at different stages of the repair reaction.
Assuntos
Reparo de Erro de Pareamento de DNA , DNA Fúngico/metabolismo , Proteínas de Ligação a DNA/fisiologia , Proteína 2 Homóloga a MutS/fisiologia , Proteínas de Saccharomyces cerevisiae/fisiologia , Saccharomyces cerevisiae/genética , Difosfato de Adenosina/metabolismo , Trifosfato de Adenosina/metabolismo , Proteínas de Ligação a DNA/química , Microscopia de Fluorescência/métodos , Modelos Moleculares , Proteína 2 Homóloga a MutS/química , Estrutura Terciária de Proteína , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/químicaRESUMO
In eukaryotic mismatch repair (MMR) MSH2-MSH6 initiates the repair of base-base and small insertion/deletion mismatches while MSH2-MSH3 repairs larger insertion/deletion mismatches. Here, we show that the msh2Delta1 mutation, containing a complete deletion of the conserved mismatch recognition domain I of MSH2, conferred a separation of function phenotype with respect to MSH2-MSH3 and MSH2-MSH6 functions. Strains bearing the msh2Delta1 mutation were nearly wild-type in MSH2-MSH6-mediated MMR and in suppressing recombination between DNA sequences predicted to form mismatches recognized by MSH2-MSH6. However, these strains were completely defective in MSH2-MSH3-mediated MMR and recombination functions. This information encouraged us to analyze the contributions of domain I to the mismatch binding specificity of MSH2-MSH3 in genetic and biochemical assays. We found that domain I in MSH2 contributed a non-specific DNA binding activity while domain I of MSH3 appeared important for mismatch binding specificity and for suppressing non-specific DNA binding. These observations reveal distinct requirements for the MSH2 DNA binding domain I in the repair of DNA mismatches and suggest that the binding of MSH2-MSH3 to mismatch DNA involves protein-DNA contacts that appear very different from those required for MSH2-MSH6 mismatch binding.
Assuntos
Pareamento Incorreto de Bases , Reparo de Erro de Pareamento de DNA , DNA Fúngico/genética , Proteínas de Ligação a DNA/genética , Proteínas Fúngicas/genética , Proteína 2 Homóloga a MutS/genética , Proteínas de Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/genética , Sequência de Aminoácidos , Sítios de Ligação , Proteínas de Ligação a DNA/metabolismo , Proteínas Fúngicas/metabolismo , Lisina/genética , Dados de Sequência Molecular , Proteína 2 Homóloga a MutS/metabolismo , Proteína 3 Homóloga a MutS , Estrutura Terciária de Proteína , Recombinação Genética , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Homologia de Sequência de Aminoácidos , Relação Estrutura-Atividade , Especificidade por SubstratoRESUMO
Genetic studies in Saccharomyces cerevisiae predict that the mismatch repair (MMR) factor MSH2-MSH3 binds and stabilizes branched recombination intermediates that form during single strand annealing and gene conversion. To test this model, we constructed a series of DNA substrates that are predicted to form during these recombination events. We show in an electrophoretic mobility shift assay that S. cerevisiae MSH2-MSH3 specifically binds branched DNA substrates containing 3' single-stranded DNA and that ATP stimulates its release from these substrates. Chemical footprinting analyses indicate that MSH2-MSH3 specifically binds at the double-strand/single-strand junction of branched substrates, alters its conformation and opens up the junction. Therefore, MSH2-MSH3 binding to its substrates creates a unique nucleoprotein structure that may signal downstream steps in repair that include interactions with MMR and nucleotide excision repair factors.
Assuntos
Reparo do DNA/genética , DNA Fúngico/química , Proteínas de Ligação a DNA/metabolismo , Proteínas Fúngicas/metabolismo , Proteína 2 Homóloga a MutS/metabolismo , Conformação de Ácido Nucleico , Recombinação Genética , Proteínas de Saccharomyces cerevisiae/metabolismo , Trifosfato de Adenosina/metabolismo , Sequência de Bases , DNA Fúngico/genética , DNA de Cadeia Simples/química , DNA de Cadeia Simples/metabolismo , Dados de Sequência Molecular , Ligação Proteica , Especificidade por SubstratoRESUMO
Mismatch repair (MMR) is initiated by MutS family proteins (MSH) that recognize DNA mismatches and recruit downstream repair factors. We used a single-molecule DNA-unzipping assay to probe interactions between S. cerevisiae MSH2-MSH6 and a variety of DNA mismatch substrates. This work revealed a high-specificity binding state of MSH proteins for mismatch DNA that was not observed in bulk assays and allowed us to measure the affinity of MSH2-MSH6 for mismatch DNA as well as its footprint on DNA surrounding the mismatch site. Unzipping analysis with mismatch substrates containing an end blocked by lac repressor allowed us to identify MSH proteins present on DNA between the mismatch and the block, presumably in an ATP-dependent sliding clamp mode. These studies provide a high-resolution approach to study MSH interactions with DNA mismatches and supply evidence to support and refute different models proposed for initiation steps in MMR.