DNA repair in the fission yeast, Schizosaccharomyces pombe

Mutants of the fission yeast Schizosaccharomyces pombe which are sensitive to UV and/or γ-irradiation have been assigned to 23 complementation groups, which can be assigned to three phenotypic groups. We have cloned genes which correct the deficiency in mutants corresponding to 12 of the complementation groups. Three genes in the excision-repair pathway have a high degree of sequence conservation with excision-repair genes from the evolutionarily distant budding yeast Saccharomyces cerevisiae. In contrast, those genes in the recombination repair pathway which have been characterised so far, show little homology with any previously characterised genes.     

Transfection of the cloned human excision repair gene ERCC-1 to UV-sensitive CHO mutants only corrects the repair defect in complementation group-2 mutants

The human DNA-excision repair gene ERCC-1 is cloned by its ability to correct the excision-repair defect of the ultraviolet light- and mitomycin-C-sensitive CHO mutant cell line 43-3B. This mutant is assigned to complementation group 2 of the excision-repair-deficient CHO mutants. In order to establish whether the correction by ERCC-1 is confined to CHO mutants of one complementation group, the cloned repair gene, present on cosmid 43-34, was transfected to representative cell lines of the 6 complementation groups that have been identified to date. Following transfection, mycophenolic acid was used to select for transferants expressing the dominant marker gene Ecogpt, also present on cosmid 43-34. Cotransfer of the ERCC-1 gene was shown by Southern blot analysis of DNA from pooled (500-2000 independent colonies) transformants of each mutant. UV survival and UV-induced UDS showed that only mutants belonging to complementation group 2 and no mutants of other groups were corrected by the ERCC-1 gene. This demonstrates that ERCC-1 does not provide an aspecific bypass of excision-repair defects in CHO mutants and supports the assumption that the complementation analysis is based on mutations in different repair genes.     

Interplay between Fanconi anemia and homologous recombination pathways in genome integrity

The Fanconi anemia (FA) pathway plays a central role in the repair of DNA interstrand crosslinks (ICLs) and regulates cellular responses to replication stress. Homologous recombination (HR), the error‐free pathway for double‐strand break (DSB) repair, is required during physiological cell cycle progression for the repair of replication‐associated DNA damage and protection of stalled replication forks. Substantial crosstalk between the two pathways has recently been unravelled, in that key HR proteins such as the RAD51 recombinase and the tumour suppressors BRCA1 and BRCA2 also play important roles in ICL repair. Consistent with this, rare patient mutations in these HR genes cause FA pathologies and have been assigned FA complementation groups. Here, we focus on the clinical and mechanistic implications of the connection between these two cancer susceptibility syndromes and on how these two molecular pathways of DNA replication and repair interact functionally to prevent genomic instability.     

Localization of the xeroderma pigmentosum group B-correcting gene ERCC3 to human chromosome 2q21

The human excision-repair gene ERCC3 was cloned after DNA-mediated gene transfer to the uv-sensitive Chinese hamster ovary mutant cell line 27-1, a member of complementation group 3 of the excision-defective rodent cell lines. The ERCC3 gene specifically corrects the DNA repair defect of xeroderma pigmentosum (XP) complementation group B, which displays the clinical symptoms of XP as well as of another rare excision-repair disorder, Cockayne syndrome. The gene encodes a presumed DNA and chromatin binding helicase, involved in early steps of the excision-repair pathway. ERCC3 was previously assigned to human chromosome 2 (L.H. Thompson, A.V. Carrano, K. Sato, E.P. Salazar, B.F. White, S.A. Stewart, J.L. Minkler, and M.J. Siciliano (1987) Somat. Cell Genet. 13: 539-551). Here we report its subchromosomal localization in the q21 region of chromosome 2 via somatic cell hybrids containing a translocated chromosome 2 and in situ hybridization with fluorescently labeled ERCC3 probes.     

Repair of 8-oxoguanine in Saccharomyces cerevisiae: interplay of DNA repair and replication mechanisms

8-Oxo-7,8-dihydroguanine (8-oxoG) is produced abundantly in DNA exposed to free radicals and reactive oxygen species. The biological relevance of 8-oxoG has been unveiled by the study of two mutator genes in Escherichia coli, fpg, and mutY. Both genes code for DNA N-glycosylases that cooperate to prevent the mutagenic effects of 8-oxoG in DNA. In Saccharomyces cerevisiae, the OGG1 gene encodes a DNA N-glycosylase/AP lyase, which is the functional homologue of the bacterial fpg gene product. The inactivation of OGG1 in yeast creates a mutator phenotype that is specific for the generation of GC to TA transversions. In yeast, nucleotide excision repair (NER) also contributes to the release of 8-oxoG in damaged DNA. Furthermore, mismatch repair (MMR) mediated by MSH2/MSH6/MLH1 plays a major role in the prevention of the mutagenic effect of 8-oxoG. Indeed, MMR acts as the functional homologue of the MutY protein of E. coli, excising the adenine incorporated opposite 8-oxoG. Finally, the efficient and accurate replication of 8-oxoG by the yeast DNA polymerase η also prevents 8-oxoG-induced mutagenesis. The aim of this review is to summarize recent literature dealing with the replication and repair of 8-oxoG in Saccharomyces cerevisiae, which can be used as a paradigm for DNA repair in eukaryotes.     

Poly(ADP-ribose) polymerase interacts with a novel human ubiquitin conjugating enzyme: hUbc9

Poly(ADP-ribose) polymerase (PARP) has been suggested to play a regulatory role in vivo, in DNA replication and/or DNA repair based mainly on its capacity to bind to DNA strand breaks. This interaction is modulated through auto poly(ADP-ribosylation). However, the biological function of PARP may also involve interactions with proteins such as topoisomerase I or DNA polymerase , which may or may not be themselves ADP-ribosylated. Using the yeast two-hybrid method search for other proteins interacting with PARP, we have isolated a full-length cDNA clone coding for a protein of 158 amino acid residues. This amino acid sequence is 66 and 56% identical to yeast ubiquitin-conjugating enzymes Hus5 and Ubc9 of Schizosaccharomyces pombe and Saccharomyces cerevisiae, respectively. Moreover, we have demonstrated that the expressed protein complements a S. cerevisiae yeast strain deficient for Ubc9. The protein encoded by the isolated cDNA is thus a new human counterpart of the ubiquitin-conjugating enzyme family and has been called hUbc9. The hubc9 gene locus has been assigned to the chromosomal location 16p13.2-p13.3. By means of two-hybrid analysis it was discovered that hUbc9 interacts with the automodification domain of PARP. This interaction was further confirmed using GST (glutathione-S-transferase) tagged fusion proteins: (i) in vivo, by transfecting cos7 cells with hUbc9 cloned in an eukaryotic expression vector, and (ii) in vitro, by mixing purified PARP with hUbc9 purified and expressed in bacteria. The possible significance and function of this interaction is discussed while taking into account the possible intracellular role of hUbc9.     

Schistosoma mansoni: the IMP4 gene is involved in DNA repair/tolerance after treatment with alkylating agent methyl methane sulfonate

Using a functional complementation strategy, we have isolated a Schistosoma mansoni cDNA that complemented Escherichia coli mutant strains which are defective in the DNA base excision repair pathway. This cDNA partially complemented the MMS-sensitive phenotype of these strains. The sequence of the isolated cDNA was homologous to genes involved in the RNA metabolism pathway, especially ScIMP4 of Saccharomyces cerevisiae. To establish whether the S. mansoni cDNA clone could complement yeast ScIMP4-defective mutants, we constructed a yeast haploid strain that coded for a truncated Imp4p protein. This mutant strain was treated with different DNA damaging agents, but showed only MMS sensitivity. The functional homology between the ScIMP4 gene and the cDNA from S. mansoni was verified by partial complementation of the mutant yeast with the worm's gene. This gene appears to be involved in DNA repair and RNA metabolism in both S. mansoni and S. cerevisiae.     

Molecular characterization of carnitine-dependent transport of acetyl-CoA from peroxisomes to mitochondria in Saccharomyces cerevisiae and identification of a plasma membrane carnitine transporter, Agp2p.

In Saccharomyces cerevisiae, beta-oxidation of fatty acids is confined to peroxisomes. The acetyl-CoA produced has to be transported from the peroxisomes via the cytoplasm to the mitochondrial matrix in order to be degraded to CO(2) and H(2)O. Two pathways for the transport of acetyl-CoA to the mitochondria have been proposed. The first involves peroxisomal conversion of acetyl-CoA into glyoxylate cycle intermediates followed by transport of these intermediates to the mitochondria. The second pathway involves peroxisomal conversion of acetyl-CoA into acetylcarnitine, which is subsequently transported to the mitochondria. Using a selective screen, we have isolated several mutants that are specifically affected in the second pathway, the carnitine-dependent acetyl-CoA transport from the peroxisomes to the mitochondria, and assigned these CDAT mutants to three different complementation groups. The corresponding genes were identified using functional complementation of the mutants with a genomic DNA library. In addition to the previously reported carnitine acetyl-CoA transferase (CAT2), we identified the genes for the yeast orthologue of the human mitochondrial carnitine acylcarnitine translocase (YOR100C or CAC) and for a transport protein (AGP2) required for carnitine transport across the plasma membrane.     

Bambi is coexpressed with Bmp-4 during mouse embryogenesis

Signaling of TGF-β superfamily members is tightly controlled by an elaborate network of regulators (for recent review see Trends Genet. 15 (1999) 3; Genes Dev. 14 (2000) 627). Recently, the transmembrane protein BAMBI (BMP and activin membrane-bound inhibitor) has been shown to interfere with Bmp and activin-like signaling by inhibiting Tgf-β type I receptor activation (Nature 401 (1999) 480). In striking contrast to other Bmp antagonists like noggin (Cell 86 (1996) 599) or chordin (Cell 86 (1996) 589), BAMBI is strictly coexpressed with Bmp-4 during early Xenopus embryogenesis. The grouping of genes according to their shared complex spatial expression pattern and their involvement in the same biological signaling pathway has been referred to as synexpression group. This concept facilitates prognoses about the roles of a group member with unknown function. Apparently, only a minority of genes is organized in synexpression groups and up to now they have mainly been described in yeast and Xenopus (for review see Nature 402 (1999) 483). In the frog, BAMBI is a member of the Bmp-4 synexpression group (Nature 401 (1999) 480). We identified two murine homologues of BAMBI one of which, named Bambi-ψ, is a pseudogene. We show that the spatiotemporal expression pattern of Bambi closely matches that of Bmp-4 during mouse embryonic development. Moreover, we show that Bambi expression is induced in mouse embryonic fibroblasts by Bmp-4. Hence, we provide first evidence for the existence of an evolutionarily conserved Bmp-4 synexpression group in mammals.     

DNA repair in reduced genome: the Mycoplasma model

The occurrence of bacteria with a reduced genome, such as that found in Mycoplasmas, raises the question as to which genes should be enough to guarantee the genomic stability indispensable for the maintenance of life. The aim of this work was to compare nine Mycoplasma genomes in regard to DNA repair genes. An in silico analysis was done using six Mycoplasma species, whose genomes are accessible at GenBank, and M. synoviae, and two strains of M. hyopneumoniae, whose genomes were recently sequenced by The Brazilian National Genome Project Consortium and Southern Genome Investigation Program (Brazil) respectively. Considering this reduced genome model, our comparative analysis suggests that the DNA integrity necessary for life can be primarily maintained by nucleotide excision repair (NER), which is the only complete repair pathway. Furthermore, some enzymes involved with base excision repair (BER) and recombination are also present and can complement the NER activity. The absence of RecR and RecO-like ORFs was observed only in M. genitalium and M. pneumoniae, which can be involved with the conservation of gene order observed between these two species. We also obtained phylogenetic evidence for the recent acquisition of the ogt gene in M. pulmonis and M. penetrans by a lateral transference event. In general, the presence or nonexistence of repair genes is shared by all species analyzed, suggesting that the loss of the majority of repair genes was an ancestral event, which occurred before the divergence of the Mycoplasma species.     

DNA excision repair in mammalian cell extracts.

The many genetic complementation groups of DNA excision-repair defective mammalian cells indicate the considerable complexity of the excision repair process. The cloning of several repair genes is taking the field a step closer to mechanistic studies of the actions and interactions of repair proteins. Early biochemical studies of mammalian DNA repair in vitro are now at hand. Repair synthesis in damaged DNA can be monitored by following the incorporation of radiolabelled nucleotides. Synthesis is carried out by mammalian cell extracts and is defective in extracts from cell lines derived from individuals with the excision-repair disorder xeroderma pigmentosum. Biochemical complementation of the defective extracts can be used to purify repair proteins. Repair of damage caused by agents including ultraviolet irradiation, psoralens, and platinating compounds has been observed. Neutralising antibodies against the human single-stranded DNA binding protein (HSSB) have demonstrated a requirement for this protein in DNA excision repair as well as in DNA replication.     

Ethanol-sensitive mutants of Saccharomyces cerevisiae

Saccharomyces cerevisiae mutants unable to grow at ethanol concentrations at which the wild type strain S288C does grow, have been isolated. Some of them show additional phenotypic alterations in colony size, temperature sensitivity and viability in ethanol, which cosegregate with the growth sensitivity in ethanol. 21 selected monogenic ethanol-sensitive mutants define 20 complementation groups, denominated ETA1 to ETA20, which indicates that there is a high number of genes involved in the ethanol tolerance/sensitivity mechanism.Out of 21 selected monogenic mutants, 20 are not altered in the glycolytic pathway since, when maintained in glucosesupplemented medium, they can produce as much ethanol as the wild type and at about the same velocity. Nor do any of the mutants seem to be altered in the lipid biosynthetic pathway since, whether grown in the absence or in the presence of ethanol, their concentration of fatty acids and ergosterol is similar to that of the wild type under the same conditions. Therefore growth sensitivity to ethanol does not seem necessarily to be related to carbohydrate or lipid metabolism.Non-common abbreviations YP yeast extract peptone medium - YPD yeast extract peptone dextrose agar or medium - YPG yeast extract peptone glycerol agar - YPDE yeast extract peptone dextrose ethanol agar or medium - SD yeast nitrogen base dextrose agar - SPO yeast extract potassium acetate glucose agar - PD parental ditype - NPD non-parental ditype - TT tetratype     

Hansenula polymorpha as a novel yeast system for the expression of heterologous genes

The advantages of Hansenula polymorpha as a new yeast expression system are discussed in terms of the powerful and regulatable methanol oxidase promoter and the organism's ability to grow on cheap carbon sources. The development of techniques for conventional genetic analysis is described. A total of 218 mutants have been assigned to 62 complementation groups, three genes have been found to be linked forming the first linkage group in this organism. Methods for molecular transformation have been developed allowing the expression of heterologous genes. The disruptive integration and expression of the neomycin phosphotransferase is described.     

Analysis of SHPRH functions in DNA repair and immunoglobulin diversification

During replication, bypass of DNA lesions is orchestrated by the Rad6 pathway. Monoubiquitination of proliferating cell nuclear antigen (PCNA) by Rad6/Rad18 leads to recruitment of translesion polymerases for direct and potentially mutagenic damage bypass. An error-free bypass pathway may be initiated via K63-linked PCNA polyubiquitination by Ubc13/Mms2 and the E3 ligase Rad5 in yeast, or HLTF/SHPRH in vertebrates. For the latter two enzymes, redundancy with a third E3 ligase and alternative functions have been reported. We have previously shown that the Rad6 pathway is involved in somatic hypermutation of immunoglobulin genes in B lymphocytes. Here, we have used knockout strategies targeting expression of the entire SHPRH protein or functionally significant domains in chicken DT40 cells that do not harbor a HLTF ortholog. We show that SHPRH is apparently redundant with another E3 ligase during DNA damage-induced PCNA modification. SHPRH plays no substantial role in cellular resistance to drugs initiating excision repair and the Rad6 pathway, but is important in survival of topoisomerase II inhibitor treatment. Removal of only the C-terminal RING domain does not interfere with this SHPRH function. SHPRH inactivation does not substantially impact on the overall efficacy of Ig diversification. Redundancy of E3 ligases in the Rad6 pathway may be linked to its different functions in genome maintenance and genetic plasticity.     

UV-specific mutations of the human patched gene in basal cell carcinomas from normal individuals and xeroderma pigmentosum patients

Germline mutations of the human patched gene, PTCH, are responsible for the nevoid basal cell carcinoma (NBCC) syndrome or Gorlin's syndrome, characterized by multiple skin cancers, internal cancers and severe developmental abnormalities. The patched gene codes for a developmental regulator protein implicated in the sonic hedgehog (SHH) signalling pathway which plays an important role in oncogenic transformation. Patched exhibits tumor suppression function and has been shown to be mutated in skin cancers isolated from DNA repair-proficient patients or from xeroderma pigmentosum (XP), a DNA repair-deficient syndrome.

We have reviewed and analyzed in detail the different mutation spectra found on the PTCH gene in these various models. The type and distribution of mutations are quite different between germline, sporadic and XP cancers. Among the germline alterations, there is a preponderance (70%) of rearrangements compared to other tumour types analysed where less than 30% of rearrangements is observed. Typical UV-induced mutations of the patched gene are found prominently in XP basal cell carcinomas (BCCs) and in particular, a significantly higher level (63%) of the UV signature tandem mutations is found compared to sporadic BCC (11%). The location of mutations along the PTCH protein delineates several important functional domains implicated in the biology of this transmembrane receptor.     

The Fanconi anemia pathway and ICL repair: implications for cancer therapy

Fanconi anemia (FA) is an inherited disease caused by mutations in at least 13 genes and characterized by genomic instability. In addition to displaying strikingly heterogenous clinical phenotypes, FA patients are exquisitely sensitive to treatments with crosslinking agents that create interstrand crosslinks (ICL). In contrast to bacteria and yeast, in which ICLs are repaired through replication-dependent and -independent mechanisms, it is thought that ICLs are repaired primarily during DNA replication in vertebrates. However, recent data indicate that replication-independent ICL repair also operates in vertebrates. While the precise role of the FA pathway in ICL repair remains elusive, increasing evidence suggests that FA proteins function at different steps in the sensing, recognition and processing of ICLs, as well as in signaling from these very toxic lesions, which can be generated by a wide variety of cancer chemotherapeutic drugs. Here, we discuss some of the recent findings that have shed light on the role of the FA pathway in ICL repair, with special emphasis on the implications of these findings for cancer therapy since disruption of FA genes have been associated with cancer predisposition.     

A Dictyostelium discoideum DNA fragment complements a Saccharomyces cerevisiae ura3 mutant

Dictyostelium discoideum DNA fragments have been inserted into the chimeric bacterium-yeast plasmid YEp13. Recombinant plasmids were used to transform yeast using a strain of Saccharomyces cerevisiae deficient in OMP decarboxylase activity. Several clones were selected for growth in uracil-free medium. One clone was further analysed and contains a plasmid with a segment of D. discoideum DNA which complements a yeast ura3 mutation.     

Isolation and characterization of Candida membranifaciens subsp. flavinogenie W14-3, a novel riboflavin-producing marine yeast

We found that the marine yeast strain W14-3 isolated from seawater of China Eastern Sea could produce riboflavin. It is interesting to observe that the marine yeast strain produced a large amount of riboflavin in the medium containing xylose, sucrose, galactose and maltose under the conditions of vigorous shaking. The yeast strain was found to belong to Candida membranifaciens subsp. flavinogenie based on the results of routine and molecular identification. The protein sequences deduced from the partial genes encoding GTP cyclohydrolase II and 3,4-dihydroxy-2-butanone-4-phosphate synthase in the yeast exhibited high identity with those of the corresponding enzymes for riboflavin biosynthesis in other yeasts. Fe³⁺ available in the medium repressed riboflavin production and expression of the genes responsible for riboflavin biosynthesis in the yeast. The results have evidenced that a riboflavin synthesis pathway indeed existed in the yeast. This is the first study to report that C. membranifaciens subsp. flavinogenie W14-3 from the marine environment could produce riboflavin.     

Genetic characterization of the pdu operon: use of 1,2-propanediol in Salmonella typhimurium.

Salmonella typhimurium is able to catabolize 1,2-propanediol for use as the sole carbon and energy source; the first enzyme of this pathway requires the cofactor adenosyl cobalamin (Ado-B12). Surprisingly, Salmonella can use propanediol as the sole carbon source only in the presence of oxygen but can synthesize Ado-B12 only anaerobically. To understand this situation, we have studied the pdu operon, which encodes proteins for propanediol degradation. A set of pdu mutants defective in aerobic degradation of propanediol (with exogenous vitamin B12) defines four distinct complementation groups. Mutations in two of these groups (pduC and pduD) eliminate propanediol dehydratase activity. Based on mutant phenotypes, a third complementation group (pduG) appears to encode a cobalamin adenosyl transferase activity. No function has been assigned to the pduJ complementation group. Propionaldehyde dehydrogenase activity is eliminated by mutations in any of the four identified complementation groups, suggesting that this activity may require a complex of proteins encoded by the operon. None of the mutations analyzed affects either of the first two genes of the operon (pduA and pduB), which were identified by DNA sequence analysis. Available data suggest that the pdu operon includes enough DNA for about 15 genes and that the four genetically identified genes are the only ones required for aerobic use of propanediol.     

Spontaneous Mitotic Recombination in Yeast: The Hyper-Recombinational Rem1 Mutations Are Alleles of the Rad3 Gene

The RAD3 gene of Saccharomyces cerevisiae is required for UV excision-repair and is essential for cell viability. We have identified the rem1 mutations (enhanced spontaneous mitotic recombination and mutation) of Saccharomyces cerevisiae as alleles of RAD3 by genetic mapping, complementation with the cloned wild-type gene, and DNA hybridization. The high levels of spontaneous mitotic gene conversion, crossing over, and mutation conferred upon cells by the rem1 mutations are distinct from the effects of all other alleles of RAD3. We present preliminary data on the localization of the rem1 mutations within the RAD3 gene. The interaction of the rem1 mutant alleles with a number of radiation-sensitive mutations is also different than the interactions reported for previously described (UV-sensitive) alleles of RAD3. Double mutants of rem1 and a defect in the recombination-repair pathway are inviable, while double mutants containing UV-sensitive alleles of RAD3 are viable. The data presented here demonstrate that: (1) rem1 strains containing additional mutations in other excision-repair genes do not exhibit elevated gene conversion; (2) triple mutants containing rem1 and mutations in both excision-repair and recombination-repair are viable; (3) such triple mutants containing rad52 have reduced levels of gene conversion but wild-type frequencies of crossing over. We have interpreted these observations in a model to explain the effects of rem1. Consistent with the predictions of the model, we find that the size of DNA from rem1 strains, as measured by neutral sucrose gradients, is smaller than wild type.