Fluorescence-based incision assay for human XPF-ERCC1 activity identifies important elements of DNA junction recognition.
Fluorescence-based incision assay for human XPF-ERCC1 activity identifies important elements of DNA junction recognition.
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DOI：
10.1093/nar/gks284
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发表时间：
2012-07
影响因子：
14.9
通讯作者：
McDonald NQ
中科院分区：
文献类型：
--
作者：
Bowles M;Lally J;Fadden AJ;Mouilleron S;Hammonds T;McDonald NQ
人XPF-ERCC 1复合物的结构特异性核酸内切酶活性对于许多有助于维持基因组完整性的DNA加工机制是必不可少的。XPF-ERCC 1切割DNA结构，如双链体的一条链中的茎环、气泡或瓣，其中至少有一条下游单链。在这里，我们定义了切割茎环底物的最低底物要求，使我们能够开发一种基于实时荧光的测定方法来测量核酸内切酶活性。使用这种检测，我们表明，在双链体的切割位点上游的序列的变化，结果在高达100倍的变化的茎环底物的切割率由XPF-ERCC 1。XPF-ERCC 1优先切割位于T或U的3′-侧的磷酸二酯键，其侧接上游T或U，表明T/U口袋可能存在于催化结构域内。除了核酸内切酶结构域和串联螺旋-发夹-螺旋结构域之外，XPF还具有发散的和无活性的DEAH解旋酶样结构域（HLD）。我们表明，删除HLD消除内切酶活性，并证明纯化的重组XPF-HLD显示出优先结合茎环结构的单链或双链体，这表明HLD在初始结构识别的作用。我们的数据一起描述了XPF-ERCC 1和公认的模型底物的特征，这些特征对于识别和有效的切口活性很重要。
The structure-specific endonuclease activity of the human XPF–ERCC1 complex is essential for a number of DNA processing mechanisms that help to maintain genomic integrity. XPF–ERCC1 cleaves DNA structures such as stem–loops, bubbles or flaps in one strand of a duplex where there is at least one downstream single strand. Here, we define the minimal substrate requirements for cleavage of stem–loop substrates allowing us to develop a real-time fluorescence-based assay to measure endonuclease activity. Using this assay, we show that changes in the sequence of the duplex upstream of the incision site results in up to 100-fold variation in cleavage rate of a stem-loop substrate by XPF-ERCC1. XPF–ERCC1 has a preference for cleaving the phosphodiester bond positioned on the 3′-side of a T or a U, which is flanked by an upstream T or U suggesting that a T/U pocket may exist within the catalytic domain. In addition to an endonuclease domain and tandem helix–hairpin–helix domains, XPF has a divergent and inactive DEAH helicase-like domain (HLD). We show that deletion of HLD eliminates endonuclease activity and demonstrate that purified recombinant XPF–HLD shows a preference for binding stem–loop structures over single strand or duplex alone, suggesting a role for the HLD in initial structure recognition. Together our data describe features of XPF–ERCC1 and an accepted model substrate that are important for recognition and efficient incision activity.
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