Postreplication DNA Repair Research Articles

Noncovalent Interaction between Ubiquitin and the Human DNA Repair Protein Mms2 Is Required for Ubc13-mediated Polyubiquitination

Ubiquitin-conjugating enzyme variants share significant sequence similarity with typical E2 (ubiquitin-conjugating) enzymes of the protein ubiquitination pathway but lack their characteristic active site cysteine residue. The MMS2 gene of Saccharomyces cerevisiae encodes one such ubiquitin-conjugating enzyme variant that is involved in the error-free DNA postreplicative repair pathway through its association with Ubc13, an E2. The Mms2-Ubc13 heterodimer is capable of linking ubiquitin molecules to one another through an isopeptide bond between the C terminus and Lys-63. Using highly purified components, we show here that the human forms of Mms2 and Ubc13 associate into a heterodimer that is stable over a range of conditions. The ubiquitin-thiol ester form of the heterodimer can be produced by the direct activation of its Ubc13 subunit with E1 (ubiquitin-activating enzyme) or by the association of Mms2 with the Ubc13-ubiquitin thiol ester. The activated heterodimer is capable of transferring its covalently bound ubiquitin to Lys-63 of an untethered ubiquitin molecule, resulting in diubiquitin as the predominant species. In (1)H (15)N HSQC ((1)H (15)N heteronuclear single quantum coherence) NMR experiments, we have mapped the surface determinants of tethered and untethered ubiquitin that interact with Mms2 and Ubc13 in both their monomeric and dimeric forms. These results have identified a surface of untethered ubiquitin that interacts with Mms2 in the monomeric and heterodimeric form. Furthermore, the C-terminal tail of ubiquitin does not participate in this interaction. These results suggest that the role of Mms2 is to correctly orient either a target-bound or untethered ubiquitin molecule such that its Lys-63 is placed proximally to the C terminus of the ubiquitin molecule that is linked to the active site of Ubc13.

Crystal structure of the human ubiquitin conjugating enzyme complex, hMms2-hUbc13.

The ubiquitin conjugating enzyme complex Mms2-Ubc13 plays a key role in post-replicative DNA repair in yeast and the NF-kappaB signal transduction pathway in humans. This complex assembles novel polyubiquitin chains onto yet uncharacterized protein targets. Here we report the crystal structure of a complex between hMms2 (Uev1) and hUbc13 at 1.85 A resolution and a structure of free hMms2 at 1.9 A resolution. These structures reveal that the hMms2 monomer undergoes a localized conformational change upon interaction with hUbc13. The nature of the interface provides a physical basis for the preference of Mms2 for Ubc13 as a partner over a variety of other structurally similar ubiquitin-conjugating enzymes. The structure of the hMms2-hUbc13 complex provides the conceptual foundation for understanding the mechanism of Lys 63 multiubiquitin chain assembly and for its interactions with the RING finger proteins Rad5 and Traf6.

DNA postreplication repair and mutagenesis in Saccharomyces cerevisiae

DNA postreplication repair (PRR) is defined as an activity to convert DNA damage-induced single-stranded gaps into large molecular weight DNA without actually removing the replication-blocking lesions. In bacteria such as Escherichia coli, this activity requires RecA and the RecA-mediated SOS response and is accomplished by recombination and mutagenic translesion DNA synthesis. Eukaryotic cells appear to share similar DNA damage tolerance pathways; however, some enzymes required for PRR in eukaryotes are rather different from those of prokaryotes. In the yeast Saccharomyces cerevisiae, PRR is centrally controlled by RAD6 and RAD18, whose products form a stable complex with single-stranded DNA-binding, ATPase and ubiquitin-conjugating activities. PRR can be further divided into translesion DNA synthesis and error-free modes, the exact molecular events of which are largely unknown. This error-free PRR is analogous to DNA damage-avoidance as defined in mammalian cells, which relies on recombination processes. Two possible mechanisms by which recombination participate in PRR to resolve the stalled replication folk are discussed. Recombination and PRR are also genetically regulated by a DNA helicase and are coupled to the cell-cycle. The PRR processes appear to be highly conserved within eukaryotes, from yeast to human.

In Vitro Assembly and Recognition of Lys-63 Polyubiquitin Chains

Polyubiquitin chains assembled through lysine 48 (Lys-48) of ubiquitin act as a signal for substrate proteolysis by 26 S proteasomes, whereas chains assembled through Lys-63 play a mechanistically undefined role in post-replicative DNA repair. We showed previously that the products of the UBC13 and MMS2 genes function in error-free post-replicative DNA repair in the yeast Saccharomyces cerevisiae and form a complex that assembles Lys-63-linked polyubiquitin chains in vitro. Here we confirm that the Mms2.Ubc13 complex functions as a high affinity heterodimer in the chain assembly reaction in vitro and report the results of a kinetic characterization of the polyubiquitin chain assembly reaction. To test whether a Lys-63-linked polyubiquitin chain can signal degradation, we conjugated Lys-63-linked tetra-ubiquitin to a model substrate of 26 S proteasomes. Although the noncanonical chain effectively signaled substrate degradation, the results of new genetic epistasis studies agree with previous genetic data in suggesting that the proteolytic activity of proteasomes is not required for error-free post-replicative repair.

Molecular Insights into Polyubiquitin Chain Assembly: Crystal Structure of the Mms2/Ubc13 Heterodimer

While the signaling properties of ubiquitin depend on the topology of polyubiquitin chains, little is known concerning the molecular basis of specificity in chain assembly and recognition. UEV/Ubc complexes have been implicated in the assembly of Lys63-linked polyubiquitin chains that act as a novel signal in postreplicative DNA repair and IκBα kinase activation. The crystal structure of the Mms2/Ubc13 heterodimer shows the active site of Ubc13 at the intersection of two channels that are potential binding sites for the two substrate ubiquitins. Mutations that destabilize the heterodimer interface confer a marked UV sensitivity, providing direct evidence that the intact heterodimer is necessary for DNA repair. Selective mutations in the channels suggest a molecular model for specificity in the assembly of Lys63-linked polyubiquitin signals.

Molecular cloning and functional characterization of two murine cDNAs which encode Ubc variants involved in DNA repair and mutagenesis

Ubiquitin-conjugating enzyme (Ubc) variants share structural similarity with Ubcs but lack the essential cysteine residue required to form a thioester bond with ubiquitin. Yeast Mms2 is a Ubc variant and plays an important role in error-free DNA postreplication repair to protect cells from killing by DNA damaging agents and mutagenesis. Ironically, one of two known Mms2 homologs, CROC1, has been linked to cell immortalization and tumorigenesis. To further investigate cellular roles played by mammalian Mms2 homologs, we report here the molecular cloning, tissue distribution and functional characterization of two mouse cDNAs encoding mMMS2 and mCROC1. Unlike human CROC1, the mCROC1 gene does not encode two alternative transcripts in most tissues. Instead, nonoverlapping sequences were found in two distinct cDNA clones that together would constitute a full-length open reading frame homologous to CROC1B. Both mMMS2 and the C-terminal mCROC1 core domain are able to complement the yeast mms2 mutant functionally and are able to interact with Ubc13 in a yeast two-hybrid assay, indicating that they are true yeast Mms2 homologs and may play a similar role in DNA postreplication repair. We propose several hypotheses to reconcile the seemingly contradictory observations regarding roles of the two mammalian Mms2 homologs in tumorigenesis and carcinogenesis.

Replication and G2 checkpoints: their response to caffeine.

Under long hydroxyurea treatments, evidence was obtained for the sequential activation of four checkpoints located between the onset of S phase and mitosis in Allium cepa L. root meristems. Biparametric flow cytometry (Br-DNA/total DNA) showed that cells initially accumulated at early S phase but, after a delay, they resumed replication and paused again at mid S phase. Cells not only overrode this second replication block but also any G2 checkpoint they encountered. Thus, a late mitotic wave was produced in the presence of hydroxyurea. The wave was formed by cells that had apparently completed their replication (normal mitoses), while others displayed anaphases/telophases with less than the expected DNA content and with chromosomal breaks (aberrant mitoses). The presence of aberrant mitoses is direct evidence for the undue override of the two G2 checkpoints responsible for surveillance of completion of DNA synthesis and repair, respectively. Caffeine selectively abrogated the G2 block produced by the checkpoint that controls post-replication DNA repair, as it advanced the entry of cells into an aberrant mitosis. However, caffeine proved not to be the universal checkpoint-evading agent as postulated. Caffeine did not modify the spontaneous override of the replication checkpoints. Moreover, it seems to enforce the checkpoint that controls the completion of DNA synthesis, as the appearance of the late wave of normal mitoses produced in the presence of hydroxyurea was prevented by the use of caffeine.

Specific aspects of the ubiquitin system in spermatogenesis.

The ubiquitin system is involved in numerous cellular processes, regulating the amounts and/or activities of specific proteins through posttranslational coupling with ubiquitin or ubiquitin-like proteins. In spermatogenesis, there appears to be a special requirement for certain components of the ubiquitin system, as exemplified in human and mouse by mutation of USP9Y and HR6B, respectively. Both genes encode proteins which take part in the ubiquitin system and are ubiquitously expressed, but their mutation generates no apparent phenotype other than male infertility. Different phases of mammalian spermatogenesis probably require different specialized activities of the ubiquitin system. It is anticipated that ubiquitination activities similar to those required during mitotic cell cycle regulation will play some role in control of the meiotic divisions. In spermatocytes, there is an intricate link among DNA repair, the ubiquitin system, and regulation of meiotic chromatin structure, as indicated by the co-localization of proteins involved in these processes on meiotic recombination complexes. HR6B and its nearly identical homolog HR6A are multiple function proteins, with ubiquitin-conjugating activity and essential roles in post-replication DNA repair. HR6B, possibly together with the ubiquitin-ligating enzyme mRAD1 8Sc, is most likely involved in chromatin re-organization during the meiotic and post-meiotic phases of spermatogenesis. Biochemical data indicate that, in particular during spermiogenesis, the general activity of the ubiquitin system is high, which most likely is related to the high requirement for massive breakdown of cytoplasmatic and nuclear proteins during this last phase of spermatogenesis.

UBC13 , a DNA-damage-inducible gene, is a member of the error-free postreplication repair pathway in Saccharomyces cerevisiae

The Ubc13 protein was recently identified for its unique role in ubiquitin (Ub) chain assembly at the Ub Lys-63 residue instead of the conventional Lys-48 residue. This activity requires Ubc13 to form a complex with Mms2 and indeed ubc13 and mms2 mutations have been shown elsewhere to be epistatic with respect to UV sensitivity. The MMS2 gene is known to be a member of the error-free DNA postreplication repair (PRR) pathway. By contrast, the Ub Lys-63 residue has been previously implicated in the error-prone PRR pathway, since yeast cells carrying the ubiK63R mutation are defective in UV-induced mutagenesis. In the present study, we attempted to define the role of UBC13 within the PRR pathway. We found that the ubc13 mutation is epistatic to mms2 and rad6, confirming that UBC13 belongs to the PRR-pathway. We also found that ubc13 is synergistic to the error-prone PRR pathway mutation rev3, indicating that UBC13 is in a pathway alternative to REV3 mutagenesis. The ubc13 mutant displays up to a 30-fold increase in the spontaneous mutation rate, and this increase is largely REV3 dependent. In addition, UV-induced mutagenesis is fully functional in the ubc13 mutant. These results together demonstrate that UBC13 is a member of the error-free PRR pathway. The involvement of UBC13 in cellular tolerance to DNA-damage is further implicated by our finding that the UBC13 transcript level is increased up to 6-fold in response to DNA-damage.

Rad6p represses yeast-hypha morphogenesis in the human fungal pathogen Candida albicans.

Rad6p plays important roles in post-replication DNA repair, chromatin organization, gene silencing and meiosis. In this study, we show that Rad6p also regulates yeast-hypha morphogenesis in the human pathogen Candida albicans. CaRAD6 gene and cDNAs were isolated and characterized revealing that the gene carries two 5'-proximal introns. CaRad6p shows a high degree of sequence similarity to Rad6 proteins from fungi to man (60-83% identity), and it suppresses the UV sensitivity and lack of induced mutagenesis displayed by a Saccharomyces cerevisiae rad6 mutant. In C. albicans, CaRAD6 expression is induced in response to UV, and CaRad6p depletion confers UV sensitivity, confirming that Rad6p serves a role in protecting this fungus against UV damage. CaRAD6 overexpression inhibits hyphal development, whereas CaRad6p depletion enhances hyphal growth. Also, CaRAD6 mRNA levels decrease during the yeast-hypha transition. These effects are dependent on Efg1p, but not Cph1p, indicating that CaRad6p acts specifically through the Efg1p morphogenetic signalling pathway to repress yeast-hypha morphogenesis.

Noncanonical MMS2-Encoded Ubiquitin-Conjugating Enzyme Functions in Assembly of Novel Polyubiquitin Chains for DNA Repair

Ubiquitin-conjugating enzyme variant (UEV) proteins resemble ubiquitin-conjugating enzymes (E2s) but lack the defining E2 active-site residue. The MMS2-encoded UEV protein has been genetically implicated in error-free postreplicative DNA repair in Saccharomyces cerevisiae. We show that Mms2p forms a specific heteromeric complex with the UBC13-encoded E2 and is required for the Ubc13p-dependent assembly of polyubiquitin chains linked through lysine 63. A ubc13 yeast strain is UV sensitive, and single, double, and triple mutants of the UBC13, MMS2, and ubiquitin (ubiK63R) genes display a comparable phenotype. These findings support a model in which an Mms2p/Ubc13p complex assembles novel polyubiquitin chains for signaling in DNA repair, and they suggest that UEV proteins may act to increase diversity and selectivity in ubiquitin conjugation.

Role of Saccharomyces cerevisiae chromatin assembly factor-I in repair of ultraviolet radiation damage in vivo.

In vitro, the protein complex Chromatin Assembly Factor-I (CAF-I) from human or yeast cells deposits histones onto DNA templates after replication. In Saccharomyces cerevisiae, the CAC1, CAC2, and CAC3 genes encode the three CAF-I subunits. Deletion of any of the three CAC genes reduces telomeric gene silencing and confers an increase in sensitivity to killing by ultraviolet (UV) radiation. We used double and triple mutants involving cac1Delta and yeast repair gene mutations to show that deletion of the CAC1 gene increases the UV sensitivity of cells mutant in genes from each of the known DNA repair epistasis groups. For example, double mutants involving cac1Delta and excision repair gene deletions rad1Delta or rad14Delta showed increased UV sensitivity, as did double mutants involving cac1Delta and deletions of members of the RAD51 recombinational repair group. cac1Delta also increased the UV sensitivity of strains with defects in either the error-prone (rev3Delta) or error-free (pol30-46) branches of RAD6-mediated postreplicative DNA repair but did not substantially increase the sensitivity of strains carrying null mutations in the RAD6 or RAD18 genes. Deletion of CAC1 also increased the UV sensitivity and rate of UV-induced mutagenesis in rad5Delta mutants, as has been observed for mutants defective in error-free postreplicative repair. Together, these data suggest that CAF-I has a role in error-free postreplicative damage repair and may also have an auxiliary role in other repair mechanisms. Like the CAC genes, RAD6 is also required for gene silencing at telomeres. We find an increased loss of telomeric gene silencing in rad6Delta cac1Delta and rad18Delta cac1Delta double mutants, suggesting that CAF-I and multiple factors in the postreplicative repair pathway influence chromosome structure.

The Fanconi anaemia group G gene FANCG is identical with XRCC9.

Fanconi anemia (FA) is an autosomal recessive disease with diverse clinical symptoms including developmental anomalies, bone marrow failure and early occurrence of malignancies. In addition to spontaneous chromosome instability, FA cells exhibit cell cycle disturbances and hypersensitivity to cross-linking agents. Eight complementation groups (A-H) have been distinguished, each group possibly representing a distinct FA gene. The genes mutated in patients of complementation groups A (FANCA; refs 4,5) and C (FANCC; ref. 6) have been identified, and FANCD has been mapped to chromosome band 3p22-26 (ref. 7). An additional FA gene has recently been mapped to chromosome 9p (ref. 8). Here we report the identification of the gene mutated in group G, FANCG, on the basis of complementation of an FA-G cell line and the presence of pathogenic mutations in four FA-G patients. We identified the gene as human XRCC9, a gene which has been shown to complement the MMC-sensitive Chinese hamster mutant UV40, and is suspected to be involved in DNA post-replication repair or cell cycle checkpoint control. The gene is localized to chromosome band 9p13 (ref. 9), corresponding with a known localization of an FA gene.

The products of the yeast MMS2 and two human homologs (hMMS2 and CROC-1) define a structurally and functionally conserved Ubc-like protein family.

Eukaryotic genes encoding ubiquitin-congugating enzyme (Ubc)-like proteins have been isolated from both human and yeast cells. The CROC-1 gene was isolated by its ability to transactivate c- fos expression in cell culture through a tandem repeat enhancer sequence. The yeast MMS2 gene was cloned by its ability to complement the methyl methanesulfonate sensitivity of the mms2-1 mutant and was later shown to be involved in DNA post-replication repair. We report here the identification of a human MMS2 ( hMMS2 ) cDNA encoding a novel human Ubc-like protein. hMMS2 and CROC-1 share >90% amino acid sequence identity, but their DNA probes hybridize to distinct transcripts. hMMS2 and CROC-1 also share approximately 50% identity and 75% similarity with the entire length of yeast Mms2. Unlike CROC-1 , whose transcript appears to be elevated in all tumor cell lines examined, the hMMS2 transcript is only elevated in some tumor cell lines. Collectively, these results indicate that eukaryotic cells may contain a highly conserved family of Ubc-like proteins that play roles in diverse cellular processes, ranging from DNA repair to signal transduction and cell differentiation. The hMMS2 and CROC-1 genes are able to functionally complement the yeast mms2 defects with regard to sensitivity to DNA damaging agents and spontaneous mutagenesis. Conversely, both MMS2 and hMMS2 were able to transactivate a c- fos - CAT reporter gene in Rat-1 cells in a transient co-transfection assay. We propose that either these proteins function in a common cellular process, such as DNA repair, or they exert their diverse biological roles through a similar biochemical interaction relative to ubiquitination.

A novel function of the DNA repair gene rhp6 in mating-type silencing by chromatin remodeling in fission yeast.

Recent studies have indicated that the DNA replication machinery is coupled to silencing of mating-type loci in the budding yeast Saccharomyces cerevisiae, and a similar silencing mechanism may operate in the distantly related yeast Schizosaccharomyces pombe. Regarding gene regulation, an important function of DNA replication may be in coupling of faithful chromatin assembly to reestablishment of the parental states of gene expression in daughter cells. We have been interested in isolating mutants that are defective in this hypothesized coupling. An S. pombe mutant fortuitously isolated from a screen for temperature-sensitive growth and silencing phenotype exhibited a novel defect in silencing that was dependent on the switching competence of the mating-type loci, a property that differentiates this mutant from other silencing mutants of S. pombe as well as of S. cerevisiae. This unique mutant phenotype defined a locus which we named sng1 (for silencing not governed). Chromatin analysis revealed a switching-dependent unfolding of the donor loci mat2P and mat3M in the sng1(-) mutant, as indicated by increased accessibility to the in vivo-expressed Escherichia coli dam methylase. Unexpectedly, cloning and sequencing identified the gene as the previously isolated DNA repair gene rhp6. RAD6, an rhp6 homolog in S. cerevisiae, is required for postreplication DNA repair and ubiquitination of histones H2A and H2B. This study implicates the Rad6/rhp6 protein in gene regulation and, more importantly, suggests that a transient window of opportunity exists to ensure the remodeling of chromatin structure during chromosome replication and recombination. We propose that the effects of the sng1(-)/rhp6(-) mutation on silencing are indirect consequences of changes in chromatin structure.

Staurosporine-sensitive protein phosphorylation is required for postreplication DNA repair in human cells

DNA repair is an important factor of stability of pro- and eukaryotic genomes which plays a central role in mutagenesis and carcinogenesis. Genetic control of nucleotide excision repair (NER) in mammalian cells is well studied, but little is known about molecular mechanisms of postreplication repair (PRR) which allows bypass of base lesions in template strands after DNA replication. In Saccharomyces cerevisiae PRR is controlled by the RAD6/RAD18 pathway which involves POL30 gene encoding proliferating cell nuclear antigen (PCNA), and in human cells PCNA is known to be closely associated with the newly replicated chromatin where PRR probably takes place. In UV-irradiated human cells distinct PCNA foci may be detected in some cells which accumulate phosphorylated breast cancer susceptibility protein BRCA1 and another protein BARD1. Human PCNA is also known to be phosphorylated after UV-irradiation. In this study we found that the known inhibitor of protein kinases staurosporine supresses PRR in NER-deficient cells which is consistent with the view that BRCA1 and PCNA are required for PRR. We also have shown that the distinct PCNA foci in UV-irradiated NER-deficient cells are actually associated with the newly replicated chromatin. Since RAD18 protein is not essential for normal DNA replication and directly controls PRR in yeast, we analysed whether this protein as well as its human homologs (HR18A and HR18B) have common domains with BRCA1 and BARD1. It is found that HR18A has a subregion of homology to BARD1 and HR18A-to BRCA1. Taken together the results indicate that BRCA1 and BARD1 may be involved in PRR in human cells.

MMS2, encoding a ubiquitin-conjugating-enzyme-like protein, is a member of the yeast error-free postreplication repair pathway.

Among the three Saccharomyces cerevisiae DNA repair epistasis groups, the RAD6 group is the most complicated and least characterized, primarily because it consists of two separate repair pathways: an error-free postreplication repair pathway, and a mutagenesis pathway. The rad6 and rad18 mutants are defective in both pathways, and the rev3 mutant affects only the mutagenesis pathway, but a yeast gene that is involved only in error-free postreplication repair has not been reported. We cloned the MMS2 gene from a yeast genomic library by functional complementation of the mms2-1 mutant [Prakash, L. & Prakash, S. (1977) Genetics 86, 33-55]. MMS2 encodes a 137-amino acid, 15.2-kDa protein with significant sequence homology to a conserved family of ubiquitin-conjugating (Ubc) proteins. However, Mms2 does not appear to possess Ubc activity. Genetic analyses indicate that the mms2 mutation is hypostatic to rad6 and rad18 but is synergistic with the rev3 mutation, and the mms2 mutant is proficient in UV-induced mutagenesis. These phenotypes are reminiscent of a pol30-46 mutant known to be impaired in postreplication repair. The mms2 mutant also displayed a REV3-dependent mutator phenotype, strongly suggesting that the MMS2 gene functions in the error-free postreplication repair pathway, parallel to the REV3 mutagenesis pathway. Furthermore, with respect to UV sensitivity, mms2 was found to be hypostatic to the rad6Delta1-9 mutation, which results in the absence of the first nine amino acids of Rad6. On the basis of these collective results, we propose that the mms2 null mutation and two other allele-specific mutations, rad6Delta1-9 and pol30-46, define the error-free mode of DNA postreplication repair, and that these mutations may enhance both spontaneous and DNA damage-induced mutagenesis.

Recombinational DNA Repair: The RecF and RecR Proteins Limit the Extension of RecA Filaments beyond Single-Strand DNA Gaps

In the presence of both the RecF and RecR proteins, RecA filament extension from a single strand gap into adjoining duplex DNA is attenuated. RecR protein alone has no effect, and RecF protein alone has a reduced activity. The RecFR complexes bind randomly, primarily to the duplex regions of the DNA, and the extension of the RecA filament is halted at the first complex encountered. A very slow lengthening of RecA filaments observed in the presence of RecFR is virtually eliminated when RecF is replaced with an RecF mutant protein that does not hydrolyze ATP. These observations are incorporated into an expanded model for the functions of RecF, RecO, and RecR proteins in the early stages of postreplication DNA repair.

Genomic instability associated with limb defects

We report a child in whom we observed markedly increased genome-wide spontaneous chromosomal breakage in both leucocytes and fibroblasts associated with severe growth retardation, radial aplasia, leucopenia, mild hydrocephalus and an unusual trichodystrophy. Exposure to DNA cross-linking agents diepoxybutane, mitomycin-C and mustine hydrochloride in this case did not result in the increased chromosome breakage seen in Fanconi anaemia. It may be that this child has a defect in postreplicative DNA repair interacting with the protein components deficient in FA.

Tobacco cotyledons: a novel system for testing mutagenicity in plants.

entiation [Dulieu and Dalebroux, 1975]. The double heterozygote can INTRODUCTION be obtained by crossing lines a1 /a1 a2/a2 , phenotypically normal and a1/a1 a2 /a2 , with partial chlorophyll deficiency. Progeny of self-polliThe study of mutagenesis and DNA repair in plants nated a1 /a1 a2 /a2 plants segregate for chlorophyll mutation alleles a1 and lags behind studies of other organisms mainly because a2 into three clearly distinguishable phenotypes with nine possible genovery few repair mutants are available [Britt, 1996]. Agrotypes. Plants a1 /a1 -/(green) and a1/a1 a2 /a2 and a1/a1 a2/a2 (pale yellow bacterium-mediated transformation and the possibility to and albino) can be easily recognised. Plants with genotypes a1 /a1 a2/a2 , a1 /a1 a2 /a2 , and a1/a1 a2 /a2 are greenish-yellow, which makes express heterologous DNA-repair genes controlled by them indistinguishable from the colour of heterozygote a1 /a1 a2 /a2 and, plant promoters can partially circumvent this drawback. as such, are evaluated in the assay. Only NTX F2 seeds were used in A quick and sensitive assay to test transgenic plants for this study. induced mutagenicity is still needed. 3. NTX tobacco transformants with the E. coli O-alkyltransferase Friedlender et al. [1996] described that cotyledon cells gene ada either under the control of mas (clone X3) [Angelis et al., 1993] or cauliflower 35S (CaMV35S) promoter (clone N1-4) [Brd iB za et can undergo three to five divisions after germination until al., 1993]. they become fully differentiated and that somatic muta4. Clones SUR9 and SUR20 of Su/su tobacco were transformed with tions can be detected in germinating seeds of chlorophyll the S. cerevisiae gene RAD6, cloned in the same expression cassette as mutants treated with mutagens. We have extended the clone N1-4 (manuscript in preparation). work of Friedlender et al. [1996] by analysis of mutagen Tobacco leaf disks were transformed via Agrobacterium-mediated TDNA transfer [Horsch et al., 1985]. Seeds of Selfed T1 plants were used sensitivity of two tobacco chlorophyll mutants and dein the cotyledon assay. Germinatiing plants segregate for the dominant rived transgenic lines: NTX clones X3 and N1-4 transphenotype acquired by T-DNA insertion (data not shown). formed with Escherichia coli gene ada coding O-alkyltransferase activity. Clone N1-4 is resistant to the toxic Cultivation and Media effect of bifunctional chloroethylating agent ACNU [Brd iB za et al., 1993], but has not yet been tested for induced Approximately 50 sterilised seeds were sown per 9-cm petri plate mutagenicity. Su/su clones SUR9 and SUR20 are transonto Murashige-Skoog media with vitamins (Duchefa, Haarlem, The formed with Saccharomyces cerevisiae gene RAD6, which has an important role in postreplication DNA repair and induced mutagenesis in yeast. Isolation of a plant Abbreviations: NTX, a double heterozygous chlorophyll mutant a1 /a1 homologue able to functionally complement yeast rad6 a2 /a2 of Nicotiana tobacum var. xanthi; MMS, methyl methanesulfonate mutant has not been reported yet. (CAS no. 66-27-3); MNU, N*-methyl-N*-nitrosourea (CAS no. 648-935); ACNU, 1-(2-chloroethyl)-1-nitroso-3-(aminomethyl-1,3-diazinnyllo)methylurea. MATERIALS AND METHODS Contract grant sponsor: Academy of Sciences of the Czech Republic; Contract grant number: A5038505; Contract grant sponsor: Czech RePlants public; Contract grant number: 204/93/1080, 521/96/0396; Contract 1. Nicotiana tabacum L. heterozygous for the Sulphur (Su) gene grant sponsor: EU Copernicus; Contract grant number: CIPA-CT94[Carlson, 1974], a nuclear gene that affects chlorophyll content in leaves. 0102. Mutations in this gene give a codominant phenotype, hence the genotype *Correspondence to: Dr. Karel J. Angelis, Institute of Experimental of Su/Su (dark green), Su/su (light green), and su/su (albino) can be Botany, Na Karlovce 1a, 160 00 Praha 6, Czech Republic; Email: codl@ easily identified. mbox.cesnet.cz. 2. A double heterozygote a1 /a1 a2 /a2 of Nicotiana tabacum var. xanthi (NTX). The two loci are homologous and involved in chloroplast differReceived 27 March 1996; revised and accepted 19 December 1996.