SummaryA genome-wide transcription pro®ling of Arabidopsis upon genotoxic stress has been performed using a high-density colony array (HDCA). The array was based on a library of 27 000 cDNA clones derived from Arabidopsis cells challenged with bleomycin plus mitomycin C. The array covers more than 10 000 individual genes (corresponding to at least 40% of Arabidopsis genes). After hybridisation of the HDCA with labelled cDNA probes obtained from genotoxin-treated (bleomycin plus mitomycin C, 6 h) and untreated seedlings, 39 genes revealed an increased and 24 genes a decreased expression among the 3200 highly expressed clones (representing approximately 1200 individual genes because of redundancy of the cDNA library). Of the 4900 clones with a low transcriptional level, the expression of 500 clones was found to be altered and 57 genes with increased and 22 genes with decreased expression were identi®ed by sequence analysis of 135 identi®ed clones. The HDCA results were validated by real-time PCR analysis. For about 80% of genes (34 out of 42), alteration in expression was con®rmed, indicating the reliability of the HDCA for transcription pro®ling. DNA damage and stress-responsive genes encoding, for instance transcription factors (myb protein and WRKY1), the ribonucleotide reductase small subunit (RNR2), thymidine kinase (TK), an AAA-type ATPase, the small subunit of a DNA polymerase and a calmodulin-like protein were found to be strongly upregulated. Also, several genes involved in cell cycle regulation revealed signi®cant alteration in transcription, as detected by real-time PCR analysis, suggesting disturbance of cell cycle progression by mutagen treatment.

The three protocols of the comet assay A/N, A/A and N/N were for the first time applied to the plant species Arabidopsis thaliana. The purpose of the experiments was to establish conditions for genotoxic exposure causing DNA damage in Arabidopsis nuclei. This is required for comprehensive gene expression profiling with the intention to screen for genes involved in response of Arabidopsis cells to genotoxic stress.Five chemicals belonging to different classes of mutagens (the monofunctional alkylating agents N-methyl-N-nitrosourea and methyl methanesulfonate, the polyfunctional alkylating agent mitomycin C, the radiomimetic bleomycin and the herbicide maleic hydrazide) were tested. Except for maleic hydrazide, dose-dependent increases in DNA damage were found using the A/N comet assay protocol. While a rapid repair of bleomycin-mediated SSBs and DSBs was found, no significant reduction of DNA migration was observed up to 48 h after treatment with the monofunctional alkylating agents.

Rad5 is the key component in the Rad5-dependent error-free branch of postreplication repair in yeast (Saccharomyces cerevisiae). Rad5 is a member of the Snf2 ATPase/helicase family, possessing as a characteristic feature, a RING-finger domain embedded in the Snf2-helicase domain and a HIRAN domain. Yeast mutants are sensitive to DNA-damaging agents and reveal differences in homologous recombination. By sequence comparisons we were able to identify two homologs (AtRAD5a and AtRAD5b) in the Arabidopsis thaliana genome, sharing about 30% identity and 45% similarity to yeast Rad5. AtRad5a and AtRad5b have the same kind of domain organization with a higher degree of similarity to each other than to ScRad5. Surprisingly, both genes differ in function: whereas two independent mutants of Atrad5a are hypersensitive to the cross-linking agents mitomycin C and cis-platin and to a lesser extent to the methylating agent, methyl methane sulfonate, the Atrad5b mutants did not exhibit any sensitivity to all DNA-damaging agents tested. An Atrad5a/Atrad5b double mutant resembles the sensitivity phenotype of the Atrad5a single mutants. Moreover, in contrast to Atrad5b, the two Atrad5a mutants are deficient in homologous recombination after treatment with the double-strand break-inducing agent bleomycin. Our results suggest that the RAD5-dependent error-free branch of postreplication repair is conserved between yeast and plants, and that AtRad5a might be functionally homologous to ScRad5.

SummaryRad17 is involved in DNA checkpoint control in yeast and human cells. A homologue of this gene as well as other genes of the pathway (the 9-1-1 complex) are present in Arabidopsis and share conserved sequence domains with their yeast and human counterparts. DNA-damaging agents induce AtRAD17 transcriptionally. AtRAD17 mutants show increased sensitivity to the DNA-damaging chemicals bleomycin and mitomycin C (MMC), which can be reversed by complementation, suggesting that the loss of function of Rad17 disturbs DNA repair in plant cells. Our results are further con®rmed by the phenotype of a mutant of the 9-1-1 complex (Rad9), which is also sensitive to the same chemicals. AtRAD9 and AtRAD17 seem to be epistatic as the double mutant is not more sensitive to the chemicals than the single mutants. The mutants show a delay in the general repair of double-strand breaks (DSBs). However, frequencies of intrachromosomal homologous recombination (HR) are enhanced. Nevertheless, the mutants are pro®cient for a further induction of HR by genotoxic stresses. Our results indicate that a mutant Rad17 pathway is associated with a general deregulation of DNA repair, which seems to be correlated with a de®ciency in non-homologous DSB repair.

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