Šamanić I, Cvitanić R, Simunić J, Puizina J
Department of Biology, Faculty of Science, University of Split, Split, Croatia.
Ruđer Bošković Institute, Zagreb, Croatia.
Plant Biol (Stuttg). 2016 Jul;18(4):681-94. doi: 10.1111/plb.12453. Epub 2016 Apr 13.
Given the fundamental role of MRE11 in many aspects of DNA metabolism and signalling in eukaryotes, we analysed the impact of several MRE11 mutations on DNA damage response (DDR) and DNA repair in Arabidopsis thaliana. Three different atmre11 and an atatm-2 mutant lines, together with the wild type (WT), were compared using a new Arabidopsis genotoxic assay for in situ evaluation of genome integrity and DNA damage repair efficiency after double strand break (DSB) induction. The results showed that, despite the phenotypic differences and different lengths of the putative truncated AtMRE11 proteins, all three atmre11 and the atatm-2 mutant lines exhibited common hypersensitivity to bleomycin treatment, where they only slightly reduced mitotic activity, indicating a G2/M checkpoint abrogation. In contrast to the WT, which reduced the frequency of chromosomal aberrations throughout the recovery period after treatment, none of the three atmre11 and atatm-2 mutants recovered. Moreover, atmre11-3 mutants, similarly to atatm-2 mutants, failed to transcriptionally induce several DDR genes and had altered expression of the CYCB1;1::GUS protein. Nevertheless, numerous chromosomal fusions in the atmre11 mutants, observed after DNA damage induction, suggest intensive DNA repair activity. These results indicate that functional and full-length AtMRE11 is essential for activation of the cell cycle arrest, transcriptional regulation and DNA repair upon induction of DSB.
鉴于MRE11在真核生物DNA代谢和信号传导的许多方面发挥着重要作用,我们分析了拟南芥中几种MRE11突变对DNA损伤反应(DDR)和DNA修复的影响。使用一种新的拟南芥遗传毒性检测方法,对三种不同的atmre11和一个atatm - 2突变体系以及野生型(WT)进行比较,以原位评估双链断裂(DSB)诱导后基因组完整性和DNA损伤修复效率。结果表明,尽管表型存在差异且假定的截短AtMRE11蛋白长度不同,但所有三种atmre11和atatm - 2突变体系对博来霉素处理均表现出共同的超敏感性,它们仅轻微降低有丝分裂活性,表明G2/M期检查点被废除。与WT不同,WT在处理后的整个恢复期降低了染色体畸变频率,而三种atmre11和atatm - 2突变体均未恢复。此外,atmre11 - 3突变体与atatm - 2突变体类似,未能转录诱导几个DDR基因,并且CYCB1;1::GUS蛋白的表达发生了改变。然而,在DNA损伤诱导后观察到的atmre11突变体中的大量染色体融合表明存在强烈的DNA修复活性。这些结果表明,功能性全长AtMRE11对于DSB诱导后的细胞周期停滞、转录调控和DNA修复的激活至关重要。