PCNA and XPF cooperate to distort DNA substrates.
PCNA and XPF cooperate to distort DNA substrates.
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DOI：
10.1093/nar/gkp1104
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发表时间：
2010-03
影响因子：
14.9
通讯作者：
Penedo JC
中科院分区：
文献类型：
--
作者：
Hutton RD;Craggs TD;White MF;Penedo JC
XPF是一种结构特异性内切酶，在各种修复过程中优先切割3‘DNA瓣。古细菌XPF蛋白与DNA双链结合的晶体结构为XPF如何识别分枝DNA结构提供了见解，最近的动力学数据表明，滑动钳PCNA作为一个重要的辅因子，可能通过允许XPF将DNA结构扭曲为合适的构象，从而进行有效的切割。在这里，我们用分子内Förster共振能量转移(FRET)研究了在有或没有增殖细胞核抗原的情况下与XPF结合的3‘-翻盖底物的溶液结构。我们证明了XPF对瓣基质的识别涉及DNA的主要构象变化，包括DNA双链的90°扭曲和单链瓣的组织。在增殖细胞核抗原的存在下，与XPF结合的膜片底物有进一步的实质性重组，为观察到增殖细胞核抗原在这一系统中具有重要的催化作用提供了结构基础。这些观察的更广泛的含义对于过量的依赖于增殖细胞核抗原的酶进行了讨论。
XPF is a structure-specific endonuclease that preferentially cleaves 3′ DNA flaps during a variety of repair processes. The crystal structure of a crenarchaeal XPF protein bound to a DNA duplex yielded insights into how XPF might recognise branched DNA structures, and recent kinetic data have demonstrated that the sliding clamp PCNA acts as an essential cofactor, possibly by allowing XPF to distort the DNA structure into a proper conformation for efficient cleavage to occur. Here, we investigate the solution structure of the 3′-flap substrate bound to XPF in the presence and absence of PCNA using intramolecular Förster resonance energy transfer (FRET). We demonstrate that recognition of the flap substrate by XPF involves major conformational changes of the DNA, including a 90° kink of the DNA duplex and organization of the single-stranded flap. In the presence of PCNA, there is a further substantial reorganization of the flap substrate bound to XPF, providing a structural basis for the observation that PCNA has an essential catalytic role in this system. The wider implications of these observations for the plethora of PCNA-dependent enzymes are discussed.
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