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.
Nucleic Acids Res. 2022 Jan 11;50(1):244-258. doi: 10.1093/nar/gkab1218.
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.
基因组稳定性的丧失会导致适应性、生育能力下降和突变率升高。因此，基因组受到监测其完整性和中和 DNA 损伤的途径的保护。为了分析胞嘧啶类似物 zebularine 诱导 DNA 损伤的机制，我们在拟南芥 zebularine 超敏结构维持染色体 6b（smc6b）突变体的背景下进行了正向抑制性遗传筛选。我们表明，smc6b 的超敏性被 EQUILIBRATIVE NUCLEOSIDE TRANSPORTER 3（ENT3）、DNA 甲基转移酶 1（MET1）和 DECREASE IN DNA METHYLATION 1（DDM1）的突变所抑制。ent3 植物对 zebularine 的抗性更强，表明 ENT3 可能是将药物导入细胞所必需的。MET1 和 DDM1 的鉴定表明，zebularine 通过干扰 CG DNA 甲基化的维持来诱导 DNA 损伤。结构类似的化合物 5-氮杂胞苷和 2-脱氧-5-氮杂胞苷也是如此。基于我们的遗传和生化数据，我们提出 zebularine 在拟南芥中诱导 MET1 和含有 zebularine 的 DNA 的酶促 DNA-蛋白质交联（DPC），这一点通过天然染色质免疫沉淀实验得到了证实。此外，zebularine 诱导的 DPC 优先在 DDM1 依赖性方式下在 45S rDNA 染色质中心积累。这些发现为研究植物中的基因组稳定性和 DPC 修复开辟了新途径。
