RESUMEN
Crossing over between homologous chromosomes is initiated in meiotic prophase in most sexually reproducing organisms by the appearance of programmed double strand breaks throughout the genome. In Saccharomyces cerevisiae the double-strand breaks are resected to form three prime single-strand tails that primarily invade complementary sequences in unbroken homologs. These invasion intermediates are converted into double Holliday junctions and then resolved into crossovers that facilitate homolog segregation during Meiosis I. Work in yeast suggests that Msh4-Msh5 stabilizes invasion intermediates and double Holliday junctions, which are resolved into crossovers in steps requiring Sgs1 helicase, Exo1, and a putative endonuclease activity encoded by the DNA mismatch repair factor Mlh1-Mlh3. We purified Mlh1-Mlh3 and showed that it is a metal-dependent and Msh2-Msh3-stimulated endonuclease that makes single-strand breaks in supercoiled DNA. These observations support a direct role for an Mlh1-Mlh3 endonuclease activity in resolving recombination intermediates and in DNA mismatch repair.
Asunto(s)
Proteínas Adaptadoras Transductoras de Señales/metabolismo , ADN Cruciforme/metabolismo , ADN de Hongos/metabolismo , Proteínas de Unión al ADN/metabolismo , Desoxirribonucleasa I/metabolismo , Meiosis/fisiología , Proteína 2 Homóloga a MutS/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/enzimología , Proteínas Adaptadoras Transductoras de Señales/genética , Roturas del ADN de Cadena Simple , ADN Cruciforme/genética , ADN de Hongos/genética , ADN Superhelicoidal/genética , ADN Superhelicoidal/metabolismo , Proteínas de Unión al ADN/genética , Desoxirribonucleasa I/genética , Homólogo 1 de la Proteína MutL , Proteínas MutL , Proteína 2 Homóloga a MutS/genética , Proteína 3 Homóloga de MutS , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/genéticaRESUMEN
Evolutionary theory assumes that mutations occur randomly in the genome; however, studies performed in a variety of organisms indicate the existence of context-dependent mutation biases. Sources of mutagenesis variation across large genomic contexts (e.g., hundreds of bases) have not been identified. Here, we use high-coverage whole-genome sequencing of a conditional mismatch repair mutant line of diploid yeast to identify mutations that accumulated after 160 generations of growth. The vast majority of the mutations accumulated as insertion/deletions (in/dels) in homopolymeric [poly(dA:dT)] and repetitive DNA tracts. Surprisingly, the likelihood of an in/del mutation in a given poly(dA:dT) tract is increased by the presence of nearby poly(dA:dT) tracts in up to a 1,000 bp region centered on the given tract. Our work suggests that specific mutation hot spots can contribute disproportionately to the genetic variation that is introduced into populations and provides long-range genomic sequence context that contributes to mutagenesis.
Asunto(s)
Mutación/genética , Poli dA-dT/genética , Saccharomyces cerevisiae/genética , Secuencia de Bases , Segregación Cromosómica/genética , Análisis por Conglomerados , Endopeptidasas/genética , Complejos de Clasificación Endosomal Requeridos para el Transporte/genética , Genoma Fúngico/genética , Genotipo , Heterocigoto , Mutación INDEL/genética , Datos de Secuencia Molecular , Mutagénesis/genética , Proteínas de Saccharomyces cerevisiae/genética , Esporas Fúngicas/genética , Ubiquitina Tiolesterasa/genéticaRESUMEN
DNA mismatch repair (MMR) models have proposed that MSH (MutS homolog) proteins identify DNA polymerase errors while interacting with the DNA replication fork. MLH (MutL homolog) proteins (primarily Mlh1-Pms1 in baker's yeast) then survey the genome for lesion-bound MSH proteins. The resulting MSH-MLH complex formed at a DNA lesion initiates downstream steps in repair. MLH proteins act as dimers and contain long (20-30 nm) unstructured arms that connect two terminal globular domains. These arms can vary between 100 and 300 amino acids in length, are highly divergent between organisms, and are resistant to amino acid substitutions. To test the roles of the linker arms in MMR, we engineered a protease cleavage site into the Mlh1 linker arm domain of baker's yeast Mlh1-Pms1. Cleavage of the Mlh1 linker arm in vitro resulted in a defect in Mlh1-Pms1 DNA binding activity, and in vivo proteolytic cleavage resulted in a complete defect in MMR. We then generated a series of truncation mutants bearing Mlh1 and Pms1 linker arms of varying lengths. This work revealed that MMR is greatly compromised when portions of the Mlh1 linker are removed, whereas repair is less sensitive to truncation of the Pms1 linker arm. Purified complexes containing truncations in Mlh1 and Pms1 linker arms were analyzed and found to have differential defects in DNA binding that also correlated with the ability to form a ternary complex with Msh2-Msh6 and mismatch DNA. These observations are consistent with the unstructured linker domains of MLH proteins providing distinct interactions with DNA during MMR.
Asunto(s)
Proteínas Adaptadoras Transductoras de Señales/química , Disparidad de Par Base/fisiología , Proteínas Portadoras/química , Reparación de la Incompatibilidad de ADN/fisiología , ADN de Hongos/metabolismo , Proteínas de Saccharomyces cerevisiae/química , Proteínas Adaptadoras Transductoras de Señales/metabolismo , Sitios de Unión , Proteínas Portadoras/metabolismo , ADN de Hongos/química , Proteínas de Unión al ADN/química , Proteínas de Unión al ADN/metabolismo , Genoma Fúngico , Homólogo 1 de la Proteína MutL , Proteínas MutL , Proteína 2 Homóloga a MutS/química , Proteína 2 Homóloga a MutS/metabolismo , Conformación Proteica , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismoRESUMEN
Design of novel DNA probes to inhibit specific repair pathways is important for basic science applications and for use as therapeutic agents. As shown previously, single pyrophosphate (PP) and O-ethyl-substituted pyrophosphate (SPP) modifications can inhibit the DNA glycosylase activities on damaged DNA. To understand the structural basis of this inhibition, the influence of the PP and SPP internucleotide groups on the helical parameters and geometry of a double-stranded DNA was studied by using molecular modeling tools including molecular dynamics and quantum mechanical-molecular mechanical (QM/MM) approaches. Native and locally modified PP- and SPP-containing DNA duplexes of dodecanucleotide d(C1G2C3G4A5A6T7T8C9G10C11G12) were simulated in aqueous solution. The energies and forces were computed by using the PBE0/6-31+G** approach in the QM part and the AMBER force-field parameters in the MM part. Analysis of the local base-pair helical parameters, internucleotide distances, and overall global structure at the located stationary points revealed a close similarity of the initial and modified duplexes, with only torsion angles of the main chain being altered in the vicinity of introduced chemical modification. Results show that the PP and SPP groups are built into a helix structure without elongation of the internucleotide distance due to flipping-out of phosphate group from the sugar-phosphate backbone. The mechanism of such embedding has only a minor impact on the base pairs stacking and Watson-Crick interactions. Biochemical studies revealed that the PP and SPP groups immediately 5', but not 3', to the 8-oxoguanosine (8oxodG) inhibit translesion synthesis by a DNA polymerase in vitro. These results suggest that subtle perturbations of the DNA backbone conformation influence processing of base lesions.
Asunto(s)
ADN/química , Difosfatos/química , ADN/efectos de los fármacos , ADN Glicosilasas/antagonistas & inhibidores , Difosfatos/farmacología , Activación Enzimática/efectos de los fármacos , Espectroscopía de Resonancia Magnética , Modelos Moleculares , Conformación de Ácido Nucleico , Sensibilidad y Especificidad , Relación Estructura-ActividadRESUMEN
Escherichia coli formamidopyrimidine-DNA glycosylase (Fpg) and human 8-oxoguanine-DNA glycosylase (hOGG1) are base excision repair enzymes involved in the 8-oxoguanine (oxoG) repair pathway. Specific contacts between these enzymes and DNA phosphate groups play a significant role in DNA-protein interactions. To reveal the phosphates crucial for lesion excision by Fpg and hOGG1, modified DNA duplexes containing pyrophosphate and OEt-substituted pyrophosphate internucleotide (SPI) groups near the oxoG were tested as substrate analogues for both proteins. We have shown that Fpg and hOGG1 recognize and specifically bind the DNA duplexes tested. We have found that both enzymes were not able to excise the oxoG residue from DNA containing modified phosphates immediately 3' to the 8-oxoguanosine (oxodG) and one nucleotide 3' away from it. In contrast, they efficiently incised DNA duplexes bearing the same phosphate modifications 5' to the oxodG and two nucleotides 3' away from the lesion. The effect of these phosphate modifications on the substrate properties of oxoG-containing DNA duplexes is discussed. Non-cleavable oxoG-containing DNA duplexes bearing pyrophosphate or SPI groups immediately 3' to the oxodG or one nucleotide 3' away from it are specific inhibitors for both 8-oxoguanine-DNA glycosylases and can be used for structural studies of complexes comprising a wild-type enzymes bound to oxoG-containing DNA.