Vonarx Edward J, Tabone Emma K, Osmond Megan J, Anderson Heather J, Kunz Bernard A
School of Life and Environmental Sciences, Deakin University, Geelong, Victoria 3217, Australia.
Plant J. 2006 May;46(3):512-21. doi: 10.1111/j.1365-313X.2006.02705.x.
Eukaryotic general transcription factor (TF) IIH is composed of 10 proteins, seven of which are also required for nucleotide excision repair (NER) of UV radiation-induced DNA damage in human cells and yeast. Plant homologues of the human TFIIH subunits XPB and XPD that function in NER have been isolated but none has been shown to operate in transcription. Here we address the capabilities of Arabidopsis thaliana AtGTF2H2 and AtXPD, homologues of the essential interacting human/yeast TFIIH components p44/Ssl1 and XPD/Rad3, respectively. Expression of AtGTF2H2 or AtXPD cDNAs in yeast ssl1 or rad3 mutants temperature-sensitive for growth due to thermolabile transcription of mRNA restored growth and so transcription at the non-permissive temperature. AtGTF2H2 also complemented the NER deficiency of the corresponding yeast mutant, as measured by full recovery of UV resistance, whereas AtXPD did not despite being necessary for NER in Arabidopsis. UV treatment did not upregulate transcription of AtGTF2H2 or AtXPD in Arabidopsis. Suppression of a yeast translation initiation defect by the ssl1-1 mutation was prevented by expression of AtGTF2H2. Deletion of SSL1 in a yeast strain expressing AtGTF2H2 did not affect growth or confer UV sensitivity, demonstrating that AtGTF2H2 can perform all essential transcription functions and UV damage repair duties of Ssl1 in its absence. Furthermore, AtGTF2H2 interacted with AtXPD and yeast Rad3, and AtXPD also interacted with yeast Ssl1 in two-hybrid assays. Our results indicate that AtGTF2H2 can act in transcription and NER, and suggest that it participates in both processes in Arabidopsis as part of TFIIH.
真核生物通用转录因子(TF)IIH由10种蛋白质组成,其中7种也是人类细胞和酵母中紫外线辐射诱导的DNA损伤核苷酸切除修复(NER)所必需的。在NER中发挥作用的人类TFIIH亚基XPB和XPD的植物同源物已被分离出来,但尚未有证据表明它们在转录中起作用。在这里,我们研究了拟南芥AtGTF2H2和AtXPD的功能,它们分别是人类/酵母TFIIH重要相互作用成分p44/Ssl1和XPD/Rad3的同源物。由于mRNA的热不稳定转录,酵母ssl1或rad3突变体在温度敏感条件下生长,AtGTF2H2或AtXPD cDNA在其中的表达恢复了生长,从而在非允许温度下恢复了转录。通过紫外线抗性的完全恢复来衡量,AtGTF2H2也弥补了相应酵母突变体的NER缺陷,而AtXPD尽管在拟南芥的NER中是必需的,但却没有。紫外线处理并未上调拟南芥中AtGTF2H2或AtXPD的转录。AtGTF2H2的表达阻止了ssl1-1突变对酵母翻译起始缺陷的抑制。在表达AtGTF2H2的酵母菌株中缺失SSL1并不影响生长或导致紫外线敏感性,这表明AtGTF2H2在没有Ssl1的情况下可以执行其所有基本的转录功能和紫外线损伤修复任务。此外,在双杂交试验中,AtGTF2H2与AtXPD和酵母Rad3相互作用,AtXPD也与酵母Ssl1相互作用。我们的结果表明AtGTF2H2可以在转录和NER中发挥作用,并表明它作为TFIIH的一部分参与了拟南芥中的这两个过程。