Algasaier Sana I, Finger L David, Bennet Ian A, Grasby Jane A
Centre for Chemical Biology, Department of Chemistry, Krebs Institute , University of Sheffield , Sheffield S3 7HF , U.K.
Biochemistry. 2018 Dec 18;57(50):6838-6847. doi: 10.1021/acs.biochem.8b00891. Epub 2018 Dec 5.
Flap endonuclease 1 (FEN1) is a structure-selective nuclease best known for its roles in the penultimate steps of Okazaki fragment maturation, long-patch base excision repair and ribonucleotide excision repair. To better understand the role of FEN1 in genome maintenance in yeast and mammals, FEN1 active site mutations (A159V and E160D) have been used as tools to dissect its involvement in DNA metabolic pathways. However, discrepancies concerning the biochemistry and molecular etiology of genomic instability when FEN1 function is altered exist. Here, a detailed biochemical and biophysical characterization of mouse FEN1 and mutants is presented. Kinetic measurements showed that the active site mutants A159V and E160D reduce the rates of hydrolysis under multiple- and single-turnover conditions on all substrates. Consistent with their dominant negative effects in heterozygotes, neither mutation affects the adoption of the substrate duplex arms in the bent conformation on the enzyme surface, although decreases in substrate binding affinity are observed. The ability of the mutants to induce the requisite local DNA conformational change near the scissile phosphate is adversely affected, suggesting that the ability to place the scissile phosphate optimally in the active site causes the reduction in rates of phosphate diester hydrolysis. Further analysis suggests that the A159V mutation causes the chemistry of phosphate diester hydrolysis to become rate-limiting, whereas the wild-type and E160D proteins are likely rate-limited by a conformational change. On the basis of these results, the proposed roles of FEN1 in genome maintenance derived from studies involving these mutations are reassessed.
翼瓣内切核酸酶1（FEN1）是一种结构选择性核酸酶，因其在冈崎片段成熟、长片段碱基切除修复和核糖核苷酸切除修复的倒数第二步中所起的作用而最为人所知。为了更好地理解FEN1在酵母和哺乳动物基因组维持中的作用，FEN1活性位点突变（A159V和E160D）已被用作剖析其参与DNA代谢途径的工具。然而，当FEN1功能改变时，关于基因组不稳定性的生物化学和分子病因学存在差异。在此，对小鼠FEN1及其突变体进行了详细的生物化学和生物物理表征。动力学测量表明，活性位点突变体A159V和E160D在多周转和单周转条件下降低了所有底物的水解速率。与其在杂合子中的显性负效应一致，尽管观察到底物结合亲和力降低，但两种突变均不影响底物双链臂在酶表面呈弯曲构象。突变体在切割磷酸酯附近诱导必要的局部DNA构象变化的能力受到不利影响，这表明将切割磷酸酯最佳地置于活性位点的能力导致磷酸二酯水解速率降低。进一步分析表明，A159V突变导致磷酸二酯水解的化学反应成为限速步骤，而野生型和E160D蛋白可能受构象变化的限速。基于这些结果，对涉及这些突变的研究所推导的FEN1在基因组维持中的作用进行了重新评估。
