Centre for Biomolecular Sciences and School of Physics and Astronomy, University of St Andrews, North Haugh, St Andrews, KY16 9SS, UK.
Nucleic Acids Res. 2010 Mar;38(5):1664-75. doi: 10.1093/nar/gkp1104. Epub 2009 Dec 11.
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 degrees 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.
XPF 是一种结构特异性内切酶，在多种修复过程中优先切割 3' DNA 发夹。与 DNA 双链结合的古菌 XPF 蛋白的晶体结构提供了有关 XPF 如何识别分支 DNA 结构的见解，最近的动力学数据表明滑动夹 PCNA 作为必需的辅助因子发挥作用，可能通过允许 XPF 扭曲 DNA 结构使其处于有效切割的适当构象。在这里，我们使用分子内Förster 共振能量转移（FRET）研究了存在和不存在 PCNA 时与 XPF 结合的 3'-发夹底物的溶液结构。我们证明 XPF 对 flap 底物的识别涉及 DNA 的主要构象变化，包括 DNA 双链的 90 度扭曲和单链 flap 的组织。在 PCNA 的存在下，与 XPF 结合的 flap 底物进一步发生实质性重组，为观察到 PCNA 在该系统中具有必需的催化作用提供了结构基础。讨论了这些观察结果对大量依赖 PCNA 的酶的广泛影响。
