Osakabe Keishi, Abe Kiyomi, Yoshioka Toji, Osakabe Yuriko, Todoriki Setsuko, Ichikawa Hiroaki, Hohn Barbara, Toki Seiichi
Plant Genetic Engineering Research Unit, Division of Plant Sciences, National Institute of Agrobiological Sciences, 2-1-2 Kannondai, Tsukuba, Ibaraki 305-8602, Japan.
Plant J. 2006 Dec;48(6):827-42. doi: 10.1111/j.1365-313X.2006.02927.x.
Homologous recombination (HR) is an essential process in maintaining genome integrity and variability. In eukaryotes, the Rad52 epistasis group proteins are involved in meiotic recombination and/or HR repair. One member of this group, Rad54, belongs to the SWI2/SNF2 family of DNA-stimulated ATPases. Recent studies indicate that Rad54 has important functions in HR, both as a chromatin remodelling factor and as a mediator of the Rad51 nucleoprotein filament. Despite the importance of Rad54 in HR, no study of Rad54 from plants has yet been performed. Here, we cloned the full-length AtRAD54 cDNA sequence; an open reading frame of 910 amino acids encodes a protein with a predicted molecular mass of 101.9 kDa. Western blotting analysis showed that the AtRad54 protein was indeed expressed as a protein of approximately 110 kDa in Arabidopsis. The predicted protein sequence of AtRAD54 contains seven helicase domains, which are conserved in all other Rad54s. Yeast two-hybrid analysis revealed an interaction between Arabidopsis Rad51 and Rad54. AtRAD54 transcripts were found in all tissues examined, with the highest levels of expression in flower buds. Expression of AtRAD54 was induced by gamma-irradiation. A T-DNA insertion mutant of AtRAD54 devoid of full-length AtRAD54 expression was viable and fertile; however, it showed increased sensitivity to gamma-irradiation and the cross-linking reagent cisplatin. In addition, the efficiency of somatic HR in the mutant plants was reduced relative to that in wild-type plants. Our findings point to an important role for Rad54 in HR repair in higher plants.
同源重组（HR）是维持基因组完整性和变异性的一个重要过程。在真核生物中，Rad52上位性组蛋白参与减数分裂重组和/或HR修复。该组的一个成员Rad54属于DNA刺激的ATP酶的SWI2/SNF2家族。最近的研究表明，Rad54在HR中具有重要功能，既是一种染色质重塑因子，也是Rad51核蛋白丝的介质。尽管Rad54在HR中很重要，但尚未对植物中的Rad54进行研究。在这里，我们克隆了全长AtRAD54 cDNA序列；一个910个氨基酸的开放阅读框编码一个预测分子量为101.9 kDa的蛋白质。蛋白质印迹分析表明，AtRad54蛋白在拟南芥中确实表达为一种约110 kDa的蛋白质。AtRAD54的预测蛋白质序列包含七个解旋酶结构域，这些结构域在所有其他Rad54中都是保守的。酵母双杂交分析揭示了拟南芥Rad51和Rad54之间的相互作用。在所有检测的组织中都发现了AtRAD54转录本，在花芽中的表达水平最高。AtRAD54的表达受到γ射线照射的诱导。一个缺乏全长AtRAD54表达的AtRAD54 T-DNA插入突变体是有活力和可育的；然而，它对γ射线照射和交联剂顺铂表现出更高的敏感性。此外，突变体植物中体细胞HR的效率相对于野生型植物有所降低。我们的研究结果表明Rad54在高等植物的HR修复中具有重要作用。
