The tomato DDI2, a PCNA ortholog, associating with DDB1-CUL4 complex is required for UV-damaged DNA repair and plant tolerance to UV stress

CULLIN 4 (CUL4)-DAMAGED DNA binding protein 1 (DDB1)-based ubiquitin E3 ligase modulates diverse cellular processes including repair of damaged genomic DNA. In this study, an uncharacterized gene termed as DDB1-Interacting protein 2 (DDI2) was identified in yeast two-hybrid screening with bait gene DDB1. The co-immunoprecipitation (co-IP) assays further demonstrated that DDI2 is associated with tomato DDB1-CUL4 complex in vivo. It appears that DDI2 encodes an ortholog of proliferating cell nuclear antigen (PCNA). Confocal microscope observation indicated that DDI2-GFP fusion protein was localized in nuclei. The expression of DDI2 gene is constitutive but substantially enhanced by UV-C irradiation. The transgenic tomato plants with overexpression or knockdown of DDI2 gene displayed the increased or decreased tolerance, respectively, to UV-C stress and chemical mutagen cisplatin. The quantitative analysis of UV-induced DNA lesions indicated that the dark repair of DNA damage was accelerated in DD12 overexpression lines but delayed in knockdown lines. Conclusively, tomato DD12 gene is required for UV-induced DNA damage repair and plant tolerance to UV stress. In addition, fruits of DD12 transgenic plants are indistinguishable from that of wild type, regarding fresh weight and nutrient quality. Therefore, overexpression of DDI2 offers a suitable strategy for genetic manipulation of enhancing plant tolerance to UV stress. (C) 2015 Elsevier Ireland Ltd. All rights reserved.

基于CULLIN 4（CUL4） - 损伤DNA结合蛋白1（DDB1）的泛素E3连接酶调节多种细胞过程，包括受损基因组DNA的修复。在本研究中，通过以DDB1为诱饵基因进行酵母双杂交筛选，鉴定出一个未被表征的基因，称为DDB1相互作用蛋白2（DDI2）。免疫共沉淀（co - IP）实验进一步证明DDI2在体内与番茄DDB1 - CUL4复合物相关联。DDI2似乎编码增殖细胞核抗原（PCNA）的同源物。共聚焦显微镜观察表明DDI2 - GFP融合蛋白定位于细胞核。DDI2基因的表达是组成型的，但受UV - C照射显著增强。DDI2基因过表达或敲低的转基因番茄植株分别对UV - C胁迫和化学诱变剂顺铂表现出耐受性增强或降低。对紫外线诱导的DNA损伤的定量分析表明，DNA损伤的暗修复在DDI2过表达株系中加速，但在敲低株系中延迟。总之，番茄DDI2基因是紫外线诱导的DNA损伤修复和植物对紫外线胁迫耐受性所必需的。此外，就鲜重和营养品质而言，DDI2转基因植株的果实与野生型无明显差异。因此，DDI2的过表达为增强植物对紫外线胁迫耐受性的基因操作提供了一种合适的策略。（C）2015爱思唯尔爱尔兰有限公司。保留所有权利。