Department of Oncology and Metabolism, Academic Unit of Molecular Oncology, University of Sheffield, Beech Hill Rd., Sheffield S10 2RX, UK.
York Structural Biology Laboratory, Department of Chemistry, University of York, York YO10 5DD, UK.
Nucleic Acids Res. 2019 Apr 8;47(6):3208-3222. doi: 10.1093/nar/gkz028.
Pif1 is a multifunctional helicase and DNA processing enzyme that has roles in genome stability. The enzyme is conserved in eukaryotes and also found in some prokaryotes. The functions of human PIF1 (hPIF1) are also critical for survival of certain tumour cell lines during replication stress, making it an important target for cancer therapy. Crystal structures of hPIF1 presented here explore structural events along the chemical reaction coordinate of ATP hydrolysis at an unprecedented level of detail. The structures for the apo as well as the ground and transition states reveal conformational adjustments in defined protein segments that can trigger larger domain movements required for helicase action. Comparisons with the structures of yeast and bacterial Pif1 reveal a conserved ssDNA binding channel in hPIF1 that we show is critical for single-stranded DNA binding during unwinding, but not the binding of G quadruplex DNA. Mutational analysis suggests that while the ssDNA-binding channel is important for helicase activity, it is not used in DNA annealing. Structural differences, in particular in the DNA strand separation wedge region, highlight significant evolutionary divergence of the human PIF1 protein from bacterial and yeast orthologues.
Pif1 是一种多功能解旋酶和 DNA 加工酶，在基因组稳定性中发挥作用。该酶在真核生物中保守，也存在于一些原核生物中。人类 PIF1（hPIF1）的功能对于复制应激期间某些肿瘤细胞系的存活也至关重要，使其成为癌症治疗的重要靶点。本文呈现的 hPIF1 晶体结构以空前的详细程度探索了沿着 ATP 水解化学反应坐标的结构事件。apo 状态以及基态和过渡态的结构揭示了在定义的蛋白质片段中发生的构象调整，这些调整可以引发解旋酶作用所需的更大的结构域运动。与酵母和细菌 Pif1 的结构比较揭示了 hPIF1 中保守的单链 DNA 结合通道，我们表明该通道对于解旋过程中单链 DNA 的结合至关重要，但对于 G 四链体 DNA 的结合则不重要。突变分析表明，虽然 ssDNA 结合通道对于解旋酶活性很重要，但它不用于 DNA 退火。结构差异，特别是在 DNA 链分离楔形区域，突出了人类 PIF1 蛋白与细菌和酵母同源物的显著进化分歧。
