Cheng Kaiying, Xu Hong, Chen Xuanyi, Wang Liangyan, Tian Bing, Zhao Ye, Hua Yuejin
Key Laboratory of Chinese Ministry of Agriculture for Nuclear-Agricultural Sciences, Institute of Nuclear-Agricultural Sciences, Zhejiang University, Hangzhou, China.
Elife. 2016 Apr 8;5:e14294. doi: 10.7554/eLife.14294.
The resection of DNA strand with a 5´ end at double-strand breaks is an essential step in recombinational DNA repair. RecJ, a member of DHH family proteins, is the only 5´ nuclease involved in the RecF recombination pathway. Here, we report the crystal structures of Deinococcus radiodurans RecJ in complex with deoxythymidine monophosphate (dTMP), ssDNA, the C-terminal region of single-stranded DNA-binding protein (SSB-Ct) and a mechanistic insight into the RecF pathway. A terminal 5´-phosphate-binding pocket above the active site determines the 5´-3´ polarity of the deoxy-exonuclease of RecJ; a helical gateway at the entrance to the active site admits ssDNA only; and the continuous stacking interactions between protein and nine nucleotides ensure the processive end resection. The active site of RecJ in the N-terminal domain contains two divalent cations that coordinate the nucleophilic water. The ssDNA makes a 180° turn at the scissile phosphate. The C-terminal domain of RecJ binds the SSB-Ct, which explains how RecJ and SSB work together to efficiently process broken DNA ends for homologous recombination.
在双链断裂处切除具有5´端的DNA链是重组DNA修复中的一个关键步骤。RecJ是DHH家族蛋白的成员之一，是参与RecF重组途径的唯一5´核酸酶。在此，我们报道了嗜放射栖热菌RecJ与脱氧胸苷单磷酸（dTMP）、单链DNA、单链DNA结合蛋白的C末端区域（SSB-Ct）形成复合物的晶体结构，并对RecF途径进行了机制性解析。活性位点上方的一个末端5´磷酸结合口袋决定了RecJ脱氧外切核酸酶的5´-3´极性；活性位点入口处的一个螺旋通道仅允许单链DNA进入；蛋白质与九个核苷酸之间连续的堆积相互作用确保了连续的末端切除。RecJ N末端结构域中的活性位点含有两个二价阳离子，它们与亲核水配位。单链DNA在可切割磷酸处发生180°转折。RecJ的C末端结构域与SSB-Ct结合，这解释了RecJ和SSB如何协同工作以有效地处理断裂的DNA末端用于同源重组。
