RESUMEN
Nucleotide-excision repair (NER) and mismatch repair (MMR) are prominent examples of highly conserved DNA repair systems which recognize and replace damaged and/or mispaired nucleotides in DNA. In humans, inheritable defects in components of the NER system are associated with severe diseases such as xeroderma pigmentosum (XP) and Cockayne syndrome (CS), whereas inactivation of MMR is accompanied by predisposition to certain types of cancer. In Schizosaccharomyces pombe, the msh2- and pms1-dependent long-patch MMR system efficiently corrects small insertion/deletion loops and all base-base mismatches, except C/C. Up to 70% of C/C mismatches generated in recombination intermediates, and to a lesser extent also other base-base mismatches, are thought to undergo correction by a minor, short-patch excision repair system. We identify here the NER genes rhpl4, swi10 and rad16 as components of this repair pathway and show that they act independently of msh2 and pms1.
Asunto(s)
Adenosina Trifosfatasas , Disparidad de Par Base , Proteínas Portadoras , Reparación del ADN/fisiología , Proteínas de Unión al ADN/genética , Proteínas Fúngicas/genética , Proteínas de Saccharomyces cerevisiae , Proteínas de Schizosaccharomyces pombe , Schizosaccharomyces/genética , Cruzamientos Genéticos , Mitosis , Datos de Secuencia Molecular , Proteína 2 Homóloga a MutS , Mutación , Recombinación Genética , Schizosaccharomyces/fisiologíaRESUMEN
Blood occlusions in the retinal microvasculature contribute to the pathology of many disease states within the eye. These events can cause haemorrhaging and retinal detachment, leading to a loss of vision in the affected patient. Here, we present a physical approach to characterising the collective cell dynamics leading to plug formation, through the use of a bespoke microfluidic device, and through the derivation of a probabilistic model. Our microfluidic device is based on a filtration design that can tune the particle volume fraction of a flowing suspension within a conduit, with sizes similar to arterioles. This allows us to control and reproduce an occlusive event. The formation of the occlusion can be examined through the extracted motion of particles within the channel, which enables the assessment of individual and collective particle dynamics in the time leading to the clogging event. In particular, we observe that at the onset of the occlusion, particles form an arch bridging the channel walls. The data presented here inform the development of our mathematical model, which captures the essential factors promoting occlusions, and notably highlights the central role of adhesion in these processes. Both the physical and probabilistic models rely on significant approximations, and future investigation will seek to assess these approximations, including the deformability and complex flow profiles of the blood constituents. However, we anticipate that the general mechanisms of occlusion may be elucidated from these simple models. As microvascular flows in the eye can now be measured in vivo and non-invasively with single cell resolution, our model will also be compared to the pathophysiological characteristics of the human microcirculation.
Asunto(s)
Microcirculación/fisiología , Microfluídica , Microvasos/fisiología , Modelos Teóricos , Oclusión de la Arteria Retiniana/fisiopatología , Oclusión de la Vena Retiniana/fisiopatología , Vasos Retinianos/fisiología , Humanos , Flujo Sanguíneo Regional/fisiologíaRESUMEN
We have identified in the fission yeast Schizosaccharomyces pombe a MutS homolog that shows highest homology to the Msh2 subgroup. msh2 disruption gives rise to increased mitotic mutation rates and increased levels of postmeiotic segregation of genetic markers. In bandshift assays performed with msh2Delta cell extracts, a general mismatch-binding activity is absent. By complementation assays, we showed that S. pombe msh2 is allelic with the previously identified swi8 and mut3 genes, which are involved in mating-type switching. The swi8-137 mutant has a mutation in the msh2 gene which causes a truncated Msh2 peptide lacking a putative DNA-binding domain. Cytological analysis revealed that during meiotic prophase of msh2-defective cells, chromosomal structures were frequently formed; such structures are rarely found in the wild type. Our data show that besides having a function in mismatch repair, S. pombe msh2 is required for correct termination of copy synthesis during mating-type switching as well as for proper organization of chromosomes during meiosis.
Asunto(s)
Disparidad de Par Base , Cromosomas Fúngicos , Reparación del ADN , Proteínas de Unión al ADN/genética , Proteínas Fúngicas , Proteínas de Schizosaccharomyces pombe , Schizosaccharomyces/genética , Alelos , Secuencia de Bases , Clonación Molecular , ADN de Hongos , Genes Fúngicos , Genes del Tipo Sexual de los Hongos , Meiosis , Mitosis , Datos de Secuencia Molecular , Proteína 2 Homóloga a MutS , Conformación de Ácido Nucleico , Profase , Esporas FúngicasRESUMEN
Repair of mismatched DNA occurs mainly by the long-patch mismatch repair (MMR) pathway, requiring Msh2 and Pms1. In Schizosaccharomyces pombe mismatches can be repaired by a short-patch repair system, containing nucleotide excision repair (NER) factors. We studied mismatch correction efficiency in cells with inactivated DNA repair nucleases Rad13, Rad2 or Uve1 in MMR proficient and deficient background. Rad13 incises 3' of damaged DNA during NER. Rad2 has a function in the Uve1-dependent repair of DNA damages and in replication. Loss of Rad13 caused a strong reduction of short-patch processing of mismatches formed during meiotic recombination. Mitotic mutation rates were increased, but not to the same extent as in the NER mutant swi10, which is defective in 5' incision. The difference might be caused by an additional role of Rad13 in base excision repair or due to partial redundancy with other 3' endonucleases. Meiotic mismatch repair was not or only slightly affected in rad2 and uve1 mutants. In addition, inactivation of uve1 caused only weak effects on mutation avoidance. Mutation rates were elevated when rad2 was mutated, but not further increased in swi10 rad2 and rad13 rad2 double mutants, indicating an epistatic relationship. However, the mutation spectra of rad2 were different from that of swi10 and rad13. Thus, the function of Rad2 in mutation avoidance is rather independent of NER. rad13, swi10 and rad2, but not uve1 mutants were sensitive to the DNA-damaging agent methyl methane sulphonate. Cell survival was further reduced in the double mutants swi10 rad2, rad13 rad2 and, surprisingly, swi10 rad13. These data confirm that NER and Rad2 act in distinct damage repair pathways and further indicate that the function of Rad13 in repair of alkylated bases is partially independent of NER.
Asunto(s)
Disparidad de Par Base/genética , Reparación del ADN/genética , Endodesoxirribonucleasas/metabolismo , Schizosaccharomyces/genética , Disparidad de Par Base/efectos de los fármacos , Cruzamientos Genéticos , Daño del ADN/efectos de los fármacos , Análisis Mutacional de ADN , Proteínas de Unión al ADN/genética , Proteínas de Unión al ADN/metabolismo , Endodesoxirribonucleasas/genética , Epistasis Genética , Proteínas Fúngicas/genética , Proteínas Fúngicas/metabolismo , Genes Fúngicos/genética , Cinética , Meiosis/genética , Metilmetanosulfonato/farmacología , Mitosis/genética , Mutagénesis/efectos de los fármacos , Mutagénesis/genética , Mutación/efectos de los fármacos , Mutación/genética , Recombinación Genética/genética , Schizosaccharomyces/citología , Schizosaccharomyces/efectos de los fármacos , Proteínas de Schizosaccharomyces pombe/genética , Proteínas de Schizosaccharomyces pombe/metabolismo , Homología de Secuencia de Ácido NucleicoRESUMEN
The mismatch repair (MMR) system ensures genome integrity by removing mispaired and unpaired bases that originate during replication. A major source of mutational changes is strand slippage in repetitive DNA sequences without concomitant repair. We established a genetic assay that allows measuring the stability of GT repeats in the ade6 gene of Schizosaccharomyces pombe. In repair-proficient strains most of the repeat variations were insertions, with addition of two nucleotides being the most frequent event. GT repeats were highly destabilized in strains defective in msh2 or pms1. In these backgrounds, mainly 2-bp insertions and 2-bp deletions occurred. Surprisingly, essentially the same high mutation rate was found with mutants defective in msh6. In contrast, a defect in swi4 (a homologue of Msh3) caused only slight effects, and instability was not further increased in msh6 swi4 double mutants. Also inactivation of exo1, which encodes an exonuclease that has an MMR-dependent function in repair of base-base mismatches, caused only slightly increased repeat instability. We conclude that Msh2, Msh6, and Pms1 have an important role in preventing tract length variations in dinucleotide repeats. Exo1 and Swi4 have a minor function, which is at least partially independent of MMR.
Asunto(s)
Disparidad de Par Base , Reparación del ADN/genética , Secuencias Repetitivas de Ácidos Nucleicos , Schizosaccharomyces/genética , Secuencia de Aminoácidos , Secuencia de Bases , Reparación del ADN/fisiología , ADN de HongosRESUMEN
The msh6 mismatch repair gene of Schizosaccharomyces pombe was cloned, sequenced, and inactivated. Strains bearing all combinations of inactivated msh6, msh2, and swi4 (the S. pombe MSH3 ortholog) alleles were tested for their defects in mitotic and meiotic mismatch repair. Mitotic mutation rates were similarly increased in msh6 and msh2 mutants, both for reversion of a base-base substitution as well as of an insertion of one nucleotide in a mononucleotide run. Tetrad analysis and intragenic two-factor crosses revealed that meiotic mismatch repair was affected in msh6 to the same extent as in msh2 background. In contrast, loss of Swi4 likely did not cause a defect in mismatch repair, but rather resulted in reduced recombination frequency. Consistently, a mutated swi4 caused a two- to threefold reduction of recombinants in intergenic crosses, while msh2 and msh6 mutants were not significantly different from wild type. In summary, our study showed that Msh6 plays the same important role as Msh2 in the major mismatch repair pathway of S. pombe, while Swi4 rather functions in recombination.
Asunto(s)
Disparidad de Par Base , Reparación del ADN/fisiología , Proteínas Fúngicas/fisiología , Schizosaccharomyces/genética , Secuencia de Aminoácidos , Secuencia de Bases , Cartilla de ADN , Reparación del ADN/genética , Proteínas Fúngicas/química , Proteínas Fúngicas/genética , Datos de Secuencia Molecular , Recombinación Genética , Homología de Secuencia de AminoácidoRESUMEN
The swi8+ gene of Schizosaccharomyces pombe appears to be involved in the termination step of copy synthesis during mating-type (MT) switching. Mutations in swi8 confer a general mutator phenotype and, in particular, generate specific mutations in the MT region. Sequencing of the MT cassettes of the h90 swi8-137 mutant revealed three altered sites. One is situated at the switching (smt) signal adjacent to the H1 homology box of the expression locus mat1:1. It reduces the rate of MT switching. The alteration at the smt signal arose frequently in other h90 swi8 strains and is probably caused by gene conversion in which the sequence adjacent to the H1 box of mat2:2 is used as template. This change might be generated during the process of MT switching when hybrid DNA formation is anomalously extended into the more heterologous region flanking the H1 homology box. In addition to the gene conversion at mat1:1, two mutations were found in the H3 homology boxes of the silent cassettes mat2:2 and mat3:3.
Asunto(s)
Genes Fúngicos , Genes del Tipo Sexual de los Hongos , Mutación , Péptidos/genética , Schizosaccharomyces/genética , Secuencia de Bases , Daño del ADN , ADN de Hongos , Conversión Génica , Factor de Apareamiento , Datos de Secuencia Molecular , FenotipoRESUMEN
The switching gene swi6 of Schizosaccharomyces pombe is involved in the repression of the silent mating-type loci mat2 and mat3. We have cloned the gene by functional complementation of the switching defect of the swi6-115 mutation. DNA sequence analyses revealed an open reading frame of 984 bp coding for a putative protein of 328 amino acids (aa). The isolation of a swi6 cDNA confirmed this result. Gene replacement showed that swi6 is not essential for viability. The Swi6 protein is very hydrophilic; it contains 41% charged aa. A region of 48 aa is homologous to a sequence motif found in the chromatin-associated proteins, HP1 and Polycomb (Drosophila melanogaster), M31, M32 and M33 (mouse), and the human HSM1 protein. This motif is called chromo domain (chromatin organization modifier). Our results indicate that Swi6 is a structural component of chromatin. Swi6 may have the function to compact mat2 and mat3 into a heterochromatin-like conformation which represses the transcription of these silent cassettes.
Asunto(s)
Proteínas Cromosómicas no Histona/genética , Proteínas de Drosophila , Proteínas Fúngicas/genética , Genes Fúngicos , Genes del Tipo Sexual de los Hongos , Genes de Cambio , Proteínas de Saccharomyces cerevisiae , Schizosaccharomyces/genética , Factores de Transcripción/genética , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Homólogo de la Proteína Chromobox 5 , Clonación Molecular , Intercambio Genético , Drosophila melanogaster/genética , Proteínas Fúngicas/química , Eliminación de Gen , Heterocromatina/química , Humanos , Ratones , Datos de Secuencia Molecular , Complejo Represivo Polycomb 1 , Proteínas/genética , Proteínas Represoras/genética , Mapeo Restrictivo , Alineación de Secuencia , Homología de Secuencia de Aminoácido , Factores de Transcripción/químicaRESUMEN
In Schizosaccharomyces pombe, an intrachromosomal crossover between the mating type (MT) expression locus and one of the silent donor cassettes is lethal due to the loss of the intervening L region. The region contains one essential gene, let1. This gene was cloned and sequenced. The deduced amino acid (aa) sequence of let1 shows extensive homologies with SUG1 from Saccharomyces cerevisiae. Significant homologies were also found with the human HIV transactivation modulators, MSS1 and TBP-1, as well as with subunit 4 of the mammalian 26 S protease. The data indicate that let1 is a member of a recently defined multigene family of ATPases.
Asunto(s)
Adenosina Trifosfatasas/genética , Proteínas Fúngicas/genética , Genes Fúngicos/genética , Genes Letales/genética , Genes del Tipo Sexual de los Hongos , Proteínas Represoras , Proteínas de Saccharomyces cerevisiae , Proteínas de Schizosaccharomyces pombe , Schizosaccharomyces/genética , Adenosina Trifosfatasas/clasificación , Secuencia de Aminoácidos , Secuencia de Bases , Clonación Molecular , VIH/genética , Humanos , Factor de Apareamiento , Datos de Secuencia Molecular , Péptidos/genética , Complejo de la Endopetidasa Proteasomal , Análisis de Secuencia de ADN , Homología de Secuencia de Aminoácido , Activación TranscripcionalRESUMEN
The swi4+ gene of Schizosaccharomyces pombe is involved in termination of copy-synthesis during mating-type switching. The gene was cloned by functional complementation of a swi4 mutant transformed with a genomic library. Determination of the nucleotide sequence revealed an open reading frame of 2979 nucleotides which is interrupted by a 68 bp long intron. The putative Swi4 protein shows homology to Duc-1 (human), Rep-3 (mouse), HexA (Streptococcus pneumoniae) and MutS (Salmonella typhimurium). The prokaryotic proteins are known as essential components involved in mismatch repair. A strain with a disrupted swi4+ gene was constructed and analysed with respect to the switching process. As in swi4 mutants duplications occur in the mating-type region of the swi4 (null) strain, reducing the efficiency of switching.
Asunto(s)
Adenosina Trifosfatasas , Reparación del ADN/genética , Proteínas de Unión al ADN , Proteínas de Escherichia coli , Proteínas Fúngicas/genética , Genes Fúngicos , Genes del Tipo Sexual de los Hongos , Genes de Cambio , Proteínas de Saccharomyces cerevisiae , Schizosaccharomyces/genética , Factores de Transcripción , Secuencia de Aminoácidos , Animales , Proteínas Bacterianas/química , Secuencia de Bases , Northern Blotting , Southern Blotting , Clonación Molecular , Proteínas Fúngicas/química , Humanos , Datos de Secuencia Molecular , Proteína MutS de Unión a los Apareamientos Incorrectos del ADN , Mapeo Restrictivo , Schizosaccharomyces/enzimología , Homología de Secuencia de Ácido NucleicoRESUMEN
Efficient mating-type (MT) switching in homothallic strains of Schizosaccharomyces pombe is significantly reduced if they have a mutation in any of the eleven known swi genes. The swi4 mutation causes heterothallic as well as homothallic segregants, both of which have duplications in the MT region. In contrast to homothallic strains, h+ swi4 strains yield only a few duplications. The duplications originate in the process of MT switching, presumably by mistakes in the resolution of DNA intermediates. They always consist of one cassette and one of the intervening sequences, L and K respectively. Strains with up to seven cassettes in the MT region were found. The possible modes of their origins are discussed.
Asunto(s)
Genes Fúngicos , Genes del Tipo Sexual de los Hongos , Mutación , Schizosaccharomyces/genética , Reordenamiento Génico , Yodo , Familia de Multigenes , Reproducción/genética , Mapeo RestrictivoRESUMEN
Stability of DNA largely depends on accuracy of repair mechanisms, which remove structural anomalies induced by exogenous and endogenous agents or introduced by DNA metabolism, such as replication. Most repair mechanisms include nucleolytic processing of DNA, where nucleases cleave a phosphodiester bond between a deoxyribose and a phosphate residue, thereby producing 5'-terminal phosphate and 3'-terminal hydroxyl groups. Exonucleases hydrolyse nucleotides from either the 5' or 3' end of DNA, while endonucleases incise internal sites of DNA. Flap endonucleases cleave DNA flap structures at or near the junction between single-stranded and double-stranded regions. DNA nucleases play a crucial role in mismatch repair, nucleotide excision repair, base excision repair and double-strand break repair. In addition, nucleolytic repair functions are required during replication to remove misincorporated nucleotides, Okazaki fragments and 3' tails that may be formed after repair of stalled replication forks.
Asunto(s)
Enzimas Reparadoras del ADN/metabolismo , Reparación del ADN , Desoxirribonucleasas/metabolismo , Proteínas Bacterianas/metabolismo , ADN/química , ADN/metabolismo , Daño del ADN , Replicación del ADN , Proteínas Fúngicas/metabolismo , Humanos , Conformación de Ácido NucleicoRESUMEN
We have performed band-shift assays to identify mismatch-binding proteins in cell extracts of Schizosaccharomyces pombe. By testing heteroduplex DNA containing either a T/G or a C/C mismatch, two distinct band shifts were produced in the gels. A low mobility complex was observed with the T/G substrate, while a high mobility complex was present with C/C. Further analysis of the mismatch-binding specificities revealed that the T/G binding activity also binds to T/C, C/T, T/T, T/-, A/-, C/-, G/-, G/G, A/A, A/C, A/G, G/T, G/A, and C/A substrates with varying efficiencies, but not binds to C/C. The C/C binding activity efficiently binds to C/C, T/C, C/T, C/A, A/C, C/-, and weakly also to T/T, while all other mispairs are not recognized. Protein extracts of a mutant strain, defective in the mutS homologue swi4, displayed both mismatch-binding activities. Thus, swi4 does not encode for either one of the mismatch-binding proteins.
Asunto(s)
ADN de Hongos/metabolismo , Proteínas de Unión al ADN/metabolismo , Proteínas Fúngicas/metabolismo , Ácidos Nucleicos Heterodúplex/metabolismo , Proteínas de Saccharomyces cerevisiae , Schizosaccharomyces/metabolismo , Factores de Transcripción , Secuencia de Bases , ADN de Cadena Simple/síntesis química , ADN de Cadena Simple/metabolismo , Proteínas Fúngicas/genética , Datos de Secuencia Molecular , Mutación , Polidesoxirribonucleótidos/síntesis química , Polidesoxirribonucleótidos/metabolismoRESUMEN
Besides the MutLS-like system, Schizosaccharomyces pombe has an additional pathway of mismatch repair. This minor pathway, producing short excision tracts, repairs C/C and, with lower efficiency, other mismatches also. We investigated the involvement of the exo1+, msh2+ and pms1+ genes in the two pathways. The exo1+ gene encodes a 5' to 3' exonuclease, while msh2+ and pms1+ are homologs of Escherichia coli mutS and mutL, respectively. Intragenic two-factor crosses showed that exo1+, msh2+ and pms1+ are involved in the major, but not in the C/C-correcting, pathway. Post-meiotic segregation frequencies and mitotic mutation rates in single and double mutants supported this finding. Furthermore, msh2 delta was epistatic over exo1 delta, and the ExoI enzyme is likely to be redundant with other exonucleases.
Asunto(s)
Proteínas Portadoras , Reparación del ADN/genética , Proteínas de Unión al ADN/metabolismo , Exodesoxirribonucleasas/metabolismo , Proteínas Fúngicas/metabolismo , Proteínas de Schizosaccharomyces pombe , Schizosaccharomyces/genética , Conversión Génica , Genes Fúngicos/genética , Meiosis/genética , Mitosis/genética , Proteína 2 Homóloga a MutS , Recombinación Genética , Schizosaccharomyces/metabolismoRESUMEN
The mismatch-binding activity Cmb1 of Schizosaccharomyces pombe was enriched from wild type cells, and N-terminal sequencing enabled cloning of the respective gene. The deduced amino acid sequence of cmb1(+) contains a high mobility group domain, a motif that is common to a heterogeneous family of DNA-binding proteins. In crude protein extracts of a cmb1 gene-disruption strain, specific binding to C/T, C/A, and C/Delta was abolished. Weak binding to C/C revealed the presence of a second mismatch-binding activity, Cmb2. Cmb1, enriched from S. pombe and purified from Escherichia coli, bound specifically to C/C, C/T, C/A, T/T, and C/Delta but showed little or no affinity to other mismatches and small loops. Cmb1 recognizes 1,2 GpG intrastrand cross-links, produced by the chemotherapeutic drug cisplatin, when two cytosines are opposite the cross-linked guanines but not when other bases are present. Consistently, O6-methylguanine:C but not O6-methylguanine/T lesions were bound. Thus, cytosines in mismatches and opposite chemically modified guanines are the preferred target of Cmb1 recognition. cmb1 mutant cells are more sensitive to cisplatin than wild type cells, indicating a role of Cmb1 in repair of cisplatin-induced DNA damage.
Asunto(s)
Disparidad de Par Base , Citosina/metabolismo , Proteínas de Unión al ADN/aislamiento & purificación , Proteínas de Unión al ADN/metabolismo , Guanina/metabolismo , Proteínas del Grupo de Alta Movilidad/aislamiento & purificación , Proteínas del Grupo de Alta Movilidad/metabolismo , Proteínas de Schizosaccharomyces pombe , Schizosaccharomyces/metabolismo , Secuencia de Aminoácidos , Secuencia de Bases , Cisplatino/farmacología , Clonación Molecular , Reactivos de Enlaces Cruzados/farmacología , ADN de Hongos/química , Proteínas de Unión al ADN/genética , Proteínas del Grupo de Alta Movilidad/genética , Metilnitronitrosoguanidina/farmacología , Datos de Secuencia Molecular , Schizosaccharomyces/efectos de los fármacosRESUMEN
Mating-type (MT) switching in homothallic (h90) strains of Schizosaccharomyces pombe is initiated by a DNA double-strand break (DSB) at the distal end of the expression cassette mat1. The cis-acting smt-s1 mutation C13-P11 reduces the frequency of MT switching. It is a small deletion mapping approximately 50 bp distal to the site of the DSB. From the h90 smt-s1 strain we isolated 13 mutants with a hyperspeckled iodine reaction. In these mutants the frequency of MT switching is increased. The mutations define nine different hsp genes, none of which maps in or close to the MT region. We tested one mutant of each gene for the presence of DSBs at mat1. Curiously, in none of the h90 smt-s1 hsp strains could DSBs be detected, although some sporulate nearly as efficiently as the h90 smt-n wild type. The hsp mutations show no effect in smt-0 strains; the smt-0 deletion abolishes MT switching completely. Furthermore, we tested the interaction of hsp1-1 with swi1, swi2 and swi7 mutations. hsp1-1 has no effect in swi2 strains, whereas it increases MT switching in swi7 and, to a lesser degree, in swi1 mutants.
Asunto(s)
ADN de Hongos/química , Genes Fúngicos , Genes del Tipo Sexual de los Hongos , Mutación , Schizosaccharomyces/genética , Autorradiografía , Proteínas de Ciclo Celular , Proteínas de Unión al ADN , Proteínas Fúngicas/genética , Yodo , Schizosaccharomyces/fisiología , Proteínas de Schizosaccharomyces pombe , Eliminación de Secuencia , Factores de Transcripción/genéticaRESUMEN
Rhp14 of Schizosaccharomyces pombe is homologous to human XPA and Saccharomyces cerevisiae Rad14, which act in nucleotide excision repair of DNA damages induced by ultraviolet light and chemical agents. Cells with disrupted rhp14 were highly sensitive to ultraviolet light, and epistasis analysis with swi10 (nucleotide excision repair) and rad2 (Uve1-dependent ultraviolet light damage repair pathway) revealed that Rhp14 is an important component of nucleotide excision repair for ultraviolet light-induced damages. Moreover, defective rhp14 caused instability of a GT repeat, similar to swi10 and synergistically with msh2 and exo1. Recombinant Rhp14 with an N-terminal hexahistidine tag was purified from Escherichia coli. Complementation studies with a rhp14 mutant demonstrated that the tagged Rhp14 is functional in repair of ultraviolet radiation-induced damages and in mitotic mutation avoidance. In bandshift assays, Rhp14 showed a preference to substrates with mismatched and unpaired nucleotides. Similarly, XPA bound more efficiently to C/C, A/C, and T/C mismatches than to homoduplex DNA. Our data show that mismatches and loops in DNA are substrates of nucleotide excision repair. Rhp14 is likely part of the recognition complex but alone is not sufficient for the high discrimination of nucleotide excision repair for modified DNA.
Asunto(s)
Disparidad de Par Base , Reparación del ADN , Proteínas de Unión al ADN/fisiología , Proteínas Fúngicas/fisiología , Proteínas de Unión al ARN/fisiología , Schizosaccharomyces/genética , Daño del ADN , Proteínas de Unión al ADN/aislamiento & purificación , Escherichia coli/genética , Proteínas de Unión al ARN/aislamiento & purificación , Proteínas Recombinantes/aislamiento & purificación , Proteína de la Xerodermia Pigmentosa del Grupo ARESUMEN
Plants are unique in the obligatory nature of their exposure to sunlight and consequently to ultraviolet (UV) irradiation. However, our understanding of plant DNA repair processes lags far behind the current knowledge of repair mechanisms in microbes, yeast and mammals, especially concerning the universally conserved and versatile dark repair pathway called nucleotide excision repair (NER). Here we report the isolation and functional characterization of Arabidopsis thaliana AtRAD1, which encodes the plant homologue of Saccharomyces cerevisiae RAD1, Schizosaccharomyces pombe RAD16 and human XPF, endonucleolytic enzymes involved in DNA repair and recombination processes. Our results indicate that AtRAD1 is involved in the excision of UV-induced damages, and allow us to assign, for the first time in plants, the dark repair of such DNA lesions to NER. The low efficiency of this repair mechanism, coupled to the fact that AtRAD1 is ubiquitously expressed including tissues that are not accessible to UV light, suggests that plant NER has other roles. Possible 'UV-independent' functions of NER are discussed with respect to features that are particular to plants.