RESUMEN
The Saccharomyces cerevisiae DNA polymerase delta proofreading exonuclease-defective mutation pol3-01 is known to cause high rates of accumulating mutations. The pol3-01 mutant was found to have abnormal cell cycle progression due to activation of the S phase checkpoint. Inactivation of the S phase checkpoint suppressed both the pol3-01 cell cycle progression defect and mutator phenotype, indicating that the pol3-01 mutator phenotype was dependent on the S phase damage checkpoint pathway. Epistasis analysis suggested that a portion of the pol3-01 mutator phenotype involves members of the RAD6 epistasis group that function in both error-free and error-prone repair. These results indicate that activation of a checkpoint in response to certain types of replicative defects can result in the accumulation of mutations.
Asunto(s)
Proteínas de Ciclo Celular , ADN Polimerasa III/genética , Reparación del ADN/genética , Proteínas de Saccharomyces cerevisiae , Saccharomyces cerevisiae/genética , Secuencia de Bases , ADN Polimerasa III/metabolismo , Replicación del ADN/genética , Mutación del Sistema de Lectura , Proteínas Fúngicas/genética , Eliminación de Gen , Genotipo , Datos de Secuencia Molecular , Mutagénesis/fisiología , Fenotipo , Proteínas Quinasas/genética , Proteínas Serina-Treonina Quinasas , Fase S/genéticaRESUMEN
Hereditary nonpolyposis colorectal carcinoma (HNPCC) is due primarily to inherited mutations in two mismatch repair genes, MSH2 and MLH1, whereas germ-line mutations in other mismatch repair genes are rare. We examined the frequency of germ-line msh6 mutations in a population-based series of 140 colorectal cancer patients, including 45 sporadic cases, 91 familial non-HNPCC cases, and 4 HNPCC cases. Among the 91 population-based familial non-HNPCC cases, germ-line msh6 mutations were found in 6 patients (7.1% of probands analyzed; median age at diagnosis, 61 years). These mutations included a splice site mutation, a frameshift mutation, two missense mutations that were demonstrated to be loss of function mutations, and two missense mutations for which functional studies were not possible. In contrast, germ-line msh6 mutations were not found in any of the 45 sporadic cases and the 4 HNPCC cases in the population-based series or in the second series of 58 clinic-based, primarily HNPCC families. Our data suggest that germ-line msh6 mutations predispose individuals to primarily late-onset, familial colorectal carcinomas that do not fulfill classic criteria for HNPCC.
Asunto(s)
Neoplasias Colorrectales/genética , Proteínas de Unión al ADN , Proteínas Fúngicas/genética , Mutación de Línea Germinal , Proteínas de Saccharomyces cerevisiae , Proteínas Adaptadoras Transductoras de Señales , Anciano , Proteínas Portadoras , Neoplasias Colorrectales Hereditarias sin Poliposis/genética , Heterocigoto , Humanos , Persona de Mediana Edad , Homólogo 1 de la Proteína MutL , Proteínas de Neoplasias/genética , Proteínas NuclearesRESUMEN
A combination of structural, thermodynamic, and transient kinetic data on wild-type and mutant Anabaena vegetative cell ferredoxins has been used to investigate the nature of the protein-protein interactions leading to electron transfer from reduced ferredoxin to oxidized ferredoxin:NADP+ reductase (FNR). We have determined the reduction potentials of wild-type vegetative ferredoxin, heterocyst ferredoxin, and 12 site-specific mutants at seven surface residues of vegetative ferredoxin, as well as the one- and two-electron reduction potentials of FNR, both alone and in complexes with wild-type and three mutant ferredoxins. X-ray crystallographic structure determinations have been carried out for six of the ferredoxin mutants. None of the mutants showed significant structural changes in the immediate vicinity of the [2Fe-2S] cluster, despite large decreases in electron-transfer reactivity (for E94K and S47A) and sizable increases in reduction potential (80 mV for E94K and 47 mV for S47A). Furthermore, the relatively small changes in Calpha backbone atom positions which were observed in these mutants do not correlate with the kinetic and thermodynamic properties. In sharp contrast to the S47A mutant, S47T retains electron-transfer activity, and its reduction potential is 100 mV more negative than that of the S47A mutant, implicating the importance of the hydrogen bond which exists between the side chain hydroxyl group of S47 and the side chain carboxyl oxygen of E94. Other ferredoxin mutations that alter both reduction potential and electron-transfer reactivity are E94Q, F65A, and F65I, whereas D62K, D68K, Q70K, E94D, and F65Y have reduction potentials and electron-transfer reactivity that are similar to those of wild-type ferredoxin. In electrostatic complexes with recombinant FNR, three of the kinetically impaired ferredoxin mutants, as did wild-type ferredoxin, induced large (approximately 40 mV) positive shifts in the reduction potential of the flavoprotein, thereby making electron transfer thermodynamically feasible. On the basis of these observations, we conclude that nonconservative mutations of three critical residues (S47, F65, and E94) on the surface of ferredoxin have large parallel effects on both the reduction potential and the electron-transfer reactivity of the [2Fe-2S] cluster and that the reduction potential changes are not the principal factor governing electron-transfer reactivity. Rather, the kinetic properties are most likely controlled by the specific orientations of the proteins within the transient electron-transfer complex.
Asunto(s)
Ferredoxina-NADP Reductasa/metabolismo , Ferredoxinas/metabolismo , Anabaena , Cristalografía por Rayos X , Ferredoxinas/genética , Modelos Químicos , Modelos Moleculares , Datos de Secuencia Molecular , Mutagénesis Sitio-Dirigida , Oxidación-Reducción , Conformación Proteica , Relación Estructura-ActividadRESUMEN
A total of 1,025,415 bases of nucleotide sequence, including the entire human immunoglobulin lambda gene locus has been determined. This is the largest contiguous human DNA sequence ever published. The sequence data revealed the organization of 36 potentially active V lambda gene segments, 33 pseudogene segments, and seven J lambda-C lambda gene segments. Among these 69 functional or nonfunctional V lambda gene segments, 32 were newly discovered. These V lambda gene segments are located within five gene-rich clusters and are divided into five clans based on sequence identity. Five potentially active nonimmunoglobulin genes were also detected within the lambda gene locus, and two other genes were observed in the upstream region. Sequence organization suggests that large DNA duplications diversified the germ-line repertoire of the V lambda gene segments.
Asunto(s)
Regiones Constantes de Inmunoglobulina/genética , Región de Unión de la Inmunoglobulina/genética , Región Variable de Inmunoglobulina/genética , Cadenas lambda de Inmunoglobulina/genética , Alelos , Empalme Alternativo , Secuencia de Aminoácidos , Secuencia de Bases , Mapeo Cromosómico , Clonación Molecular , Cósmidos , ADN/análisis , Análisis Mutacional de ADN , Exones , Expresión Génica , Humanos , Intrones , Datos de Secuencia Molecular , Familia de Multigenes/genética , Filogenia , Seudogenes/genética , Análisis de Secuencia de ADN , Homología de Secuencia de Ácido NucleicoRESUMEN
A series of charge reversal mutations in a highly conserved acidic patch on the surface of Anabaena ferredoxin (Fd), comprising residues D67, D68, and D69, have been constructed by site-directed mutagenesis. One such mutant, D68K, has a rate constant for electron transfer (et) to Anabaena ferredoxin:NADP+ reductase (FNR) at low ionic strength (I = 12 mM) which is 2.5 times larger than wild type (9000 vs 3600 s-1). This mutant Fd became indistinguishable from the wild-type protein in its reactivity at I > or = 100 mM. The other mutants showed various degrees of impairment in their et reactions with FNR over the entire range of ionic strengths. The degrees of such impairment for the D67K and D69K mutants were similar to that of the double mutant D67K/D69K. The double mutant D68K/ D69K had et activity intermediate between these mutants and wild type, whereas incorporation of the "super" mutation, D68K, into the double mutant, resulting in the D67K/D68K/D69K triple mutant, did not significantly alter the impairment caused by the D67K/D69K double mutation. Binding constants for complex formation (Kd) between the oxidized mutant proteins and oxidized FNR (except for that of the triple mutant which was not measurable), and the kinetically determined Kd values for the intermediate Fdred:FNRox complex, showed no correlation with et rate constants or with the extent of charge reversal. These results indicate that hydrophobic interactions play a key role in determining complex stability. They also provide strong support for the contention that the specific protein/protein geometry within the Fdred:FNRox intermediate complex is the major determinant of the et rate constants in this series of mutants, and that this is optimized largely by hydrophobic rather than electrostatic interactions. When electrostatic forces are dominant, as they are at low ionic strength, this can lead to nonoptimal et orientations.