The aging factor in health and disease: the promise of basic research on aging.
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Aging↗
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Werner syndrome: genetic and molecular basis of a premature aging disorder.
Werner syndrome (WS) is a rare autosomal recessive disorder characterized by genomic instability and by the premature onset of a number of age-related diseases, including cancers. The gene responsible for WS encodes a protein that has an exonuclease domain and a domain similar to DNA helicases of the RecQ-like subfamily. Accumulating evidence indicates that the WS gene product is involved in resolving aberrant DNA structures that may arise during the process of DNA replication and transcription. Such processes generate regions of single-stranded DNA that may inadvertently provide a substrate for the initiation of recombination. Various mechanisms have evolved to ensure that recombination does not occur promiscuously during these events, and results are consistent with a model in which the WS protein is part of one such mechanism.
Animals↗
Mitochondrial DNA mutators.
In this article we review our current knowledge of the mechanisms by which point mutations arise in the mitochondrial DNA (mtDNA) of Saccharomyces cerevisiae and discuss to what extent these mechanisms operate in human mtDNA mutagenesis. The 3'-5' exonuclease proofreading activity of Pol gamma ensures accuracy of mtDNA replication in both yeast and humans, while the role of base excision repair in mtDNA error avoidance remains debated. The mitochondrial mismatch repair Msh1 protein, which removes transitions in yeast, is absent in humans, a particularity that might cause accumulation of transitions, while the most frequent substitution in yeast mtDNA is A:T to T:A transversion. Proofreading-deficient mutator human cell lines and knockin mice have been created. They will be useful for studying the mechanisms by which mtDNA mutations accumulate in diseases, ageing, malignancy and drug therapy.
Animals↗
Human progeroid syndromes, aging and cancer: new genetic and epigenetic insights into old questions.
Disorders in which individuals exhibit certain features of aging early in life are referred to as segmental progeroid syndromes. With the progress that has been made in understanding the etiologies of these conditions in the past decade, potential therapeutic options have begun to move from the realm of improbability to initial stages of testing. Among these syndromes, relevant advances have recently been made in Werner syndrome, one of several progeroid syndromes characterized by defective DNA helicases, and Hutchinson-Gilford progeria syndrome, which is characterized by aberrant processing of the nuclear envelope protein lamin A. Although best known for their causative roles in these illnesses, Werner protein and lamin A have also recently emerged as key players vulnerable to epigenetic changes that contribute to tumorigenesis and aging. These advances further demonstrate that understanding progeroid syndromes and introducing adequate treatments will not only prove beneficial to patients suffering from these dramatic diseases, but will also provide new mechanistic insights into cancer and normal aging processes.
Aging↗
Yme2p is a mediator of nucleoid structure and number in mitochondria of the yeast Saccharomyces cerevisiae.
A large number of gene products have been identified that either directly or indirectly alter the inheritance of mitochondrial DNA. In yeast, we have used a unique genetic screen based on the transfer of DNA from mitochondria to nucleus to identify nuclear-encoded gene products that are targeted to mitochondria and impact the stable inheritance of mitochondrial DNA. A specific allele of one of these genes, yme2-4, prevents even the low wild-type rate of mitochondrial DNA transfer to the nucleus and imparts significant temperature-sensitive and respiratory-growth defects. Intra- and extragenic suppressors of the yme2-4 growth phenotypes were isolated and analysis of these interacting genes reveals that both YME2 and its suppressors influence the structure and number of mitochondrial nucleoids. The yme2-4 allele decreases the average number of mtDNA nucleoids found in cells and the sensitivity of DNA in toluene-treated mitochondria to digestion by DNA exonuclease, effects reversed by intra- and extragenic suppressors. The extragenic suppressor, a missense allele of ILV5, encodes an enzyme of the branched-chain amino acid biosynthetic pathway that is also a component of mitochondrial nucleoids. A null allele of ILV5 suppresses transfer of mitochondrial DNA to the nucleus and displays synthetic interactions with yme2-4.
Alleles↗
Requirement of S. pombe exonuclease II, a homologue of S. cerevisiae Sep1, for normal mitotic growth and viability.
Exonuclease II (ExoII) from Schizosaccharomyces pombe is a 5'-->3' single-stranded DNA exonuclease. We have cloned its gene, exo2, whose nucleotide sequence revealed that ExoII is a homologue of the multifunctional Saccharomyces cerevisiae Sep1 protein (also called Kem1, Xrn1, Rar5, Dst2). S. pombe exo2 null mutants were cold-sensitive for growth, had increased cell size at the restrictive temperature, were hypersensitive to the mitotic inhibitor thiabendazol and to caffeine, and died rapidly in stationary phase. Many of these phenotypes are similar to those of sep1 (kem1 or xrn1) mutants of S. cerevisiae. In contrast, the exo2 mutation had only a moderate effect on progression through meiosis and no significant effect on meiotic recombination. We discuss possible functions of the multifunctional ExoII protein.
Amino Acid Sequence↗
Schizosaccharomyces pombe exo1 is involved in the same mismatch repair pathway as msh2 and pms1.
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.
Carrier Proteins↗
Molecular cloning and genetic characterization of the Saccharomyces cerevisiae NGS1/MRE11 gene.
The Saccharomyces cerevisiae ngs1-1 mutant was previously identified by its enhanced sensitivity to simple DNA-alkylating agents such as methyl methanesulfonate but not to UV. Molecular cloning and sequencing of NGS1 as a putative DNA-alkylation repair gene revealed that it isidentical to MRE11, a gene that is involved in DNA recombinational repair. In order to investigate functional domains of the Mre11 protein, nucleotide-sequence alterations of a number of mre11 mutant alleles, including ngs1-1, mre11-1 (ts), mre11-2, mre11-3 and mre11-58, were determined. Most of these mutations map to the N-terminus ofMre11, emphasizing the importance of this highly conserved domain. The ngs1-1 and mre11-3 mutants carry nonsense mutations resulting in truncated proteins. Missense mutations were found in mre11-1 (ts), mre11-2 and mre11-58, of which mre11-2 and mre11-58 mapped to the conserved phosphoesterase domains, indicating the involvement of these motifs in the formation and/or processing of DNA double-strand breaks. Finally, mitotic-recombination assays show that the mre11 delta mutation enhances inter-chromosomal recombination but decreases the intra-chromosomal deletion frequency. In addition, MRE11 appears to play different roles during spontaneous and alkylation-induced homologous mitotic recombination.
Antineoplastic Agents, Alkylating↗
DNA-binding factors assemble in a sequence-specific manner on the maize mitochondrial atpA promoter.
The maize mitochondrial atpA promoter has been well-characterized using in vitro transcription. The functional elements of this promoter comprise a central domain extending from -7 to +5 relative to the transcription start site, and an upstream domain of 1-3 bp that is purine-rich and centered around positions -11 to -12. As a first step in characterizing the transcriptional machinery, exonuclease-III mapping (toeprinting) was used to map the borders of DNA-protein interactions using either a 107-bp wild-type template or transcriptionally-inactive templates containing linker-scanning mutations. These experiments revealed that, with a wild-type promoter, protein factors occupy as much as 36 bp, from positions -20 to +16 relative to the transcription initiation site. Protein-binding patterns were altered when the linker-scanning mutants were used, suggesting that either the number or conformation of DNA-binding proteins could account for their inability to promote transcription initiation.
Base Sequence↗
Quantitative trait loci analysis of heat stress resistance of spermatocytes in the MRL/MpJ mouse.
The MRL/MpJ mouse has previously been reported to possess an interesting phenotype in which spermatocytes are resistant to the abdominal temperature heat shock. In this study genetic analysis for it was performed. The phenotypes of F2 progenies produced by mating MRL/MpJ and control strain C57BL/6 mice were not segregated into two types as parental phenotypes, suggesting that the phenotype is controlled by multiple genetic loci. Thus, quantitative trait loci (QTL) analysis was performed using 98 microsatellite markers. The weight ratio of the cryptorchid testis to the intact testis (testis weight ratio) and the Sertoli cell index were used for quantitative traits. QTL analysis revealed two significant QTLs located on Chrs 1 and 11 for testis weight ratio and one significant QTL located in the same region of Chr 1 for the Sertoli cell index. A microsatellite marker locus located in the peak of the QTL on Chr 1 did not recombine with the exonuclease 1 (Exo1) gene locus in 140 F2 progenies. Mutation of the Exo1 gene was previously reported to be responsible for metaphase-specific apoptosis (MSA) of spermatocytes in the MRL/MpJ mouse. These results raise the possibility that mutation of the Exo1 gene is responsible for both MSA and heat stress resistance of spermatocytes in the MRL/MpJ mouse.
Animals↗
EXO1 and MSH4 differentially affect crossing-over and segregation.
The 5'-3' exonuclease Exo1p from Saccharomyces cerevisiae is required for wild-type levels of meiotic crossing-over and normal meiotic chromosome segregation as is the meiosis-specific MutS homologue, Msh4p. Mutations in both genes reduce crossing-over by approximately two-fold, but deltamsh4 strains have significantly lower viability and a higher frequency of meiosis I non-disjunction. Epistasis analysis indicates a complex interaction between the two genes. Although crossing-over was not detectably lower in the double mutant, viability was significantly worse than either single mutant. Such a result suggests that the two genes are affecting meiotic viability by distinct mechanisms. We propose that deltaexo1 affects chromosome segregation by reducing crossing-over, while deltamsh4 affects both the frequency and distribution of crossovers. Mutation in EXO1 reduces gene conversion frequencies significantly at some but not all loci, suggesting that other enzymes are also involved in DNA resection. We propose that Exo1p plays an early role in establishing some recombination intermediates by generating single-stranded tails. The role of Msh4p is suggested to be in determining whether some recombination intermediates are resolved as crossover events and in generating crossover interference. The synergistic effect of deltaexo1deltamsh4 on spore viability suggests that the two genes have partially compensatory roles in a process affecting meiotic success.
Alleles↗
Recombinational error and deletion formation in Neisseria gonorrhoeae: a role for RecJ in the production of pilE (L) deletions.
Genetic linkage within Neisseria gonorrhoeae populations is in equilibrium, yet the physical linkage map indicates a relatively stable chromosome structure, despite an apparently vast potential for mispairing between repeated sequences (e.g. between the multiple pil or opa alleles, or through mispairing of any of the numerous small repeated sequences that are liberally scattered throughout the chromosome). Therefore, the stability of the physical linkage map suggests that aberrant recombination between repeated sequences is a rare event. This study was undertaken to explore some of the parameters that may govern deletion events between short direct oligonucleotide repeats, using a chromosomal locus that appears to be especially prone to deletions (the pilin expression locus; pilE). In this report, we demonstrate that deletion formation at pilE occurs primarily through recombinational error following a pilE/pilS interaction; illegitimate (i.e. RecA-independent) events can occur, but they are infrequent. In contrast, when genetically engineered opa deletion substrates were constructed and placed in the chromosome, deletions at the opa loci were infrequent even under rec(+) conditions. A model is presented in which the gonococcal RecA and RecJ proteins promote pilE deletions through a recombination event that is templated or stabilised by a pilE/pilS interaction.
Bacterial Proteins↗
Enhanced expression of the DNA damage-inducible gene DIN7 results in increased mutagenesis of mitochondrial DNA in Saccharomyces cerevisiae.
We reported previously that the product of DIN7, a DNA damage-inducible gene of Saccharomyces cerevisiae, belongs to the XPG family of proteins, which are involved in DNA repair and replication. This family includes the S. cerevisiae protein Rad2p and its human homolog XPGC, Rad27p and its mammalian homolog FEN-1, and Exonuclease I (Exo I). Interestingly, Din7p is the only member of the XPG family which specifically functions in mitochondria. We reported previously that overexpression of DIN7 results in a mitochondrial mutator phenotype. In the present study we wished to test the hypothesis that this phenotype is dependent on the nuclease activity of Din7p. For this purpose, we constructed two alleles, din7-D78A and din7-D173A, which encode proteins in which highly conserved aspartates important for the nuclease activity of the XPG proteins have been replaced by alanines. Here, we report that overexpression of the mutant alleles, in contrast to DIN7, fails to increase the frequency of mitochondrial petite mutants or erythromycin-resistant (Er) mutants. Also, overproduction of din7-D78Ap does not result in destabilization of poly GT tracts in mitochondrial DNA (mtDNA), the phenotype observed in cells that overexpress Din7p. We also show that petite mutants induced by enhanced synthesis of wild-type Din7p exhibit gross rearrangements of mtDNA, and that this correlates with enhanced recombination within the mitochondrial cyt b gene. These results suggest that the stability of the mitochondrial genome of S. cerevisiae is modulated by the level of the nuclease Din7p.
Base Sequence↗
The jmjN and jmjC domains of the yeast zinc finger protein Gis1 interact with 19 proteins involved in transcription, sumoylation and DNA repair.
The jumonji domain is a highly conserved bipartite domain made up of two subdomains, jmjN and jmjC, which is found in many eukaryotic transcription factors. The jmjC domain was recently shown to possess the histone demethylase activity. Here we show that the jmjN and jmjC domains of the yeast zinc finger protein Gis1 interact in a two-hybrid system with 19 yeast proteins that include the RecQ helicase Sgs1, the silencing factors Esc1 and Sir4, the URI-type prefoldin Bud27 and the PIAS type SUMO ligase Nfi1/Siz2. Extensive interaction cross dependencies further suggest that the proteins form a larger complex. Consistent with this, 16 of the proteins also interact with a Bud27 two-hybrid bait, and three of them co-precipitate with TAP-tagged Gis1. The Gis1 jumonji domain can repress transcription when recruited to a promoter as a lexA fusion. This effect is dependent on both the jmjN and jmjC subdomains, as were all 19 two-hybrid interactions, indicating that the two subdomains form a single functional unit. The human Sgs1 homolog WRN also interacts with the Gis1 jumonji domain. Finally, we note that several jumonji domain interactors are related to proteins that are found in mammalian PML nuclear bodies.
Exodeoxyribonucleases↗
Neisseria gonorrhoeae recJ mutants show defects in recombinational repair of alkylated bases and UV-induced pyrimidine dimers.
Neisseria gonorrhoeae lacks several common DNA repair pathways found in other organisms. As recent evidence had indicated that gonococci use recombinational repair to repair UV-induced DNA lesions, this study examined whether the gonococcal RecJ homologue contributes in this repair capacity. The recJ gene from strain MS11 was cloned and sequenced and was found to show a considerable degree of identity to its Escherichia coli homologue. A N. gonorrhoeae delta recJ mutant was constructed and tested for recombinational proficiency as well as for defects in DNA repair. In the absence of the RecJ exonuclease, DNA transformation and pilin switching occurred at wild type levels, indicating that the efficiency of recombination remained unimpaired. In contrast, N. gonorrhoeae delta recJ mutants showed extreme sensitivity to low levels of UV irradiation and to exposure to DNA-alkylating reagents [e.g. ethyl methanesulfonate (EMS) and methyl methanesulfonate (MMS)]. Complementation of the gonococcal recJ mutant in cis restored resistance to low-level UV, indicating that the gonococcal RecJ protein is involved in recombinational repair, and can act independently of other single-strand-specific exonucleases. Furthermore, transformation competence was not required for RecJ-dependent DNA repair. Overall, the data show that N. gonorrhoeae recJ mutants present a unique phenotype when compared to their E. coli recJ counterparts, and further support the contention that RecORJ-dependent recombinational repair is a major DNA repair pathway in the genus Neisseria.
Alkylation↗
Characterization of a novel DNA damage-inducible gene of Saccharomyces cerevisiae, DIN7, which is a structural homolog of the RAD2 and RAD27 DNA repair genes.
A number of DNA damage-inducible genes (DIN) have been identified in Saccharomyces cerevisiae. In the present study we describe isolation of a novel gene, Din7, the expression of which is induced by exposure of cells to UV light, MMS (methyl methane-sulfonate) or HU (hydoxyurea). The DNA sequence of DIN7 was determined. By comparison of the predicted Din7 amino acid sequence with those in databases we found that it belongs to a family of proteins which includes S. cerevisiae Rad2 and its Schizosaccharomyces pombe and human homologs Rad13 and XPGC; S. cerevisiae Rad27 and its S. pombe homolog Rad2, and S. pombe Exo I. All these proteins are endowed with DNA nuclease activity and are known to play an important function in DNA repair. The strongest homology to Din7 was found with the Dhs1 protein of S. cerevisiae, the function of which is essentially unknown. The expression of the DIN7 gene was studied in detail using a DIN7-lacZ fusion integrated into a chromosome. We show that the expression level of DIN7 rises during meiosis at a time nearly coincident with commitment to recombination. No inducibility of DIN7 was found after treatment with DNA-damaging agents of cells bearing the rad53-21 mutation. Surprisingly, a high basal level of DIN7 expression was found in strains in which the DUN1 gene was inactivated by transposon insertion. We suggest that a form of Dun1 may be a negative regulator of the DIN7 gene expression.
Amino Acid Sequence↗
Budding yeast Rad50, Mre11, Xrs2, and Hdf1, but not Rad52, are involved in the formation of deletions on a dicentric plasmid.
We have previously shown that the RAD50, RAD52, MRE11, XRS2, and HDF1 genes of Saccharomyces cervisiae are involved in the formation of deletions by illegitimate recombination on a monocentric plasmid. In this study, we investigated the effects of mutations of these genes on formation of deletions of a dicentric plasmid, in which DNA double-strand breaks are expected to occur frequently because the two centromeres are pulled to opposite poles in mitosis. We transformed yeast cells with a dicentric plasmid, and after incubation for a few division cycles, cells carrying deleted plasmids were detected using negative selection markers. Deletions occurred at a higher frequency than on the monocentric plasmid and there were short regions of homology at the recombination junctions as observed on the monocentric plasmid. In rad50, mre11, xrs2, and hdf1 mutants, the frequency of occurrence of deletions was reduced by about 50-fold, while in the rad52 mutant, it was comparable to that in the wild-type strain. The end-joining functions of Rad50, Mre11, Xrs2, and Hdf1, suggest that these proteins play important roles in the joining of DNA ends produced on the dicentric plasmid during mitosis.
Base Sequence↗
G2/M checkpoint genes of Saccharomyces cerevisiae: further evidence for roles in DNA replication and/or repair.
We have cloned, sequenced and disrupted the checkpoint genes RAD17, RAD24 and MEC3 of Saccharomyces cerevisiae. Mec3p shows no strong similarity to other proteins currently in the database. Rad17p is similar to Rec1 from Ustilago maydis, a 3' to 5' DNA exonuclease/checkpoint protein, and the checkpoint protein Rad1p from Schizosaccharomyces pombe (as we previously reported). Rad24p shows sequence similarity to replication factor C (RFC) subunits, and the S. pombe Rad17p checkpoint protein, suggesting it has a role in DNA replication and/or repair. This hypothesis is supported by our genetic experiments which show that overexpression of RAD24 strongly reduces the growth rate of yeast strains that are defective in the DNA replication/repair proteins Rfc1p (cdc44), DNA pol alpha (cdc17) and DNA pol delta (cdc2) but has much weaker effects on cdc6, cdc9, cdc15 and CDC4 strains. The idea that RAD24 overexpression induces DNA damage, perhaps by interfering with replication/repair complexes, is further supported by our observation that RAD24 overexpression increases mitotic chromosome recombination in CDC4 strains. Although RAD17, RAD24 and MEC3 are not required for cell cycle arrest when S phase is inhibited by hydroxyurea (HU), they do contribute to the viability of yeast cells grown in the presence of HU, possibly because they are required for the repair of HU-induced DNA damage. In addition, all three are required for the rapid death of cdc13 rad9 mutants. All our data are consistent with models in which RAD17, RAD24 and MEC3 are coordinately required for the activity of one or more DNA repair pathways that link DNA damage to cell cycle arrest.
Alkylating Agents↗