Loss of genome stability leads to reduced fitness, fertility and a high mutation rate. Therefore, the genome is guarded by the pathways monitoring its integrity and neutralizing DNA lesions. To analyze the mechanism of DNA damage induction by cytidine analog zebularine, we performed a forward-directed suppressor genetic screen in the background of Arabidopsis thaliana zebularine-hypersensitive structural maintenance of chromosomes 6b (smc6b) mutant. We show that smc6b hypersensitivity was suppressed by the mutations in EQUILIBRATIVE NUCLEOSIDE TRANSPORTER 3 (ENT3), DNA METHYLTRANSFERASE 1 (MET1) and DECREASE IN DNA METHYLATION 1 (DDM1). Superior resistance of ent3 plants to zebularine indicated that ENT3 is likely necessary for the import of the drug to the cells. Identification of MET1 and DDM1 suggested that zebularine induces DNA damage by interference with the maintenance of CG DNA methylation. The same holds for structurally similar compounds 5-azacytidine and 2-deoxy-5-azacytidine. Based on our genetic and biochemical data, we propose that zebularine induces enzymatic DNA-protein crosslinks (DPCs) of MET1 and zebularine-containing DNA in Arabidopsis, which was confirmed by native chromatin immunoprecipitation experiments. Moreover, zebularine-induced DPCs accumulate preferentially in 45S rDNA chromocenters in a DDM1-dependent manner. These findings open a new avenue for studying genome stability and DPC repair in plants.

Department of Botany Faculty of Science Palacký University Olomouc Šlechtitelů 27 78371 Olomouc Czech Republic

Department of Cell Biology and Genetics Faculty of Science Palacký University Šlechtitelů 27 78371 Olomouc Czech Republic

Department of Cell Biology Centre of the Region Haná for Biotechnological and Agricultural Research Faculty of Science Palacký University Olomouc Šlechtitelů 27 78371 Olomouc Czech Republic

Institute of Experimental Botany The Czech Academy of Sciences Centre of the Region Haná for Biotechnological and Agricultural Research Šlechtitelů 31 77900 Olomouc Czech Republic

Max Planck Institute for Plant Breeding Research Carl von Linné Weg 10 50829 Cologne Germany

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