Wu X, Li J, Li X, Hsieh C L, Burgers P M, Lieber M R
Department of Pathology, Washington University School of Medicine, St Louis, MO 63110, USA.
Nucleic Acids Res. 1996 Jun 1;24(11):2036-43. doi: 10.1093/nar/24.11.2036.
In eukaryotic cells, a 5' flap DNA endonuclease activity and a ds DNA 5'-exonuclease activity exist within a single enzyme called FEN-1 [flap endo-nuclease and 5(five)'-exo-nuclease]. This 42 kDa endo-/exonuclease, FEN-1, is highly homologous to human XP-G, Saccharomyces cerevisiae RAD2 and S.cerevisiae RTH1. These structure-specific nucleases recognize and cleave a branched DNA structure called a DNA flap, and its derivative called a pseudo Y-structure. FEN-1 is essential for lagging strand DNA synthesis in Okazaki fragment joining. FEN-1 also appears to be important in mismatch repair. Here we find that human PCNA, the processivity factor for eukaryotic polymerases, physically associates with human FEN-1 and stimulates its endonucleolytic activity at branched DNA structures and its exonucleolytic activity at nick and gap structures. Structural requirements for FEN-1 and PCNA loading provide an interesting picture of this stimulation. PCNA loads on to substrates at double-stranded DNA ends. In contrast, FEN-1 requires a free single-stranded 5' terminus and appears to load by tracking along the single-stranded DNA branch. These physical constraints define the range of DNA replication, recombination and repair processes in which this family of structure-specific nucleases participate. A model explaining the exonucleolytic activity of FEN-1 in terms of its endonucleolytic activity is proposed based on these observations.
在真核细胞中，一种5' 瓣状DNA内切核酸酶活性和双链DNA 5'-外切核酸酶活性存在于一种名为FEN-1[瓣状内切核酸酶和5（五）'-外切核酸酶]的单一酶中。这种42 kDa的内切/外切核酸酶FEN-1与人类XP-G、酿酒酵母RAD2和酿酒酵母RTH1高度同源。这些结构特异性核酸酶识别并切割一种称为DNA瓣的分支DNA结构及其衍生物伪Y结构。FEN-1在冈崎片段连接的滞后链DNA合成中至关重要。FEN-1在错配修复中似乎也很重要。在这里，我们发现真核生物聚合酶的持续合成因子人类增殖细胞核抗原（PCNA）与人类FEN-1发生物理结合，并刺激其在分支DNA结构处的内切核酸酶活性以及在切口和缺口结构处的外切核酸酶活性。FEN-1和PCNA加载的结构要求为这种刺激提供了一幅有趣的图景。PCNA在双链DNA末端加载到底物上。相比之下，FEN-1需要一个游离的单链5' 末端，并且似乎通过沿着单链DNA分支追踪来加载。这些物理限制定义了这一结构特异性核酸酶家族参与的DNA复制、重组和修复过程的范围。基于这些观察结果，提出了一个根据FEN-1的内切核酸酶活性来解释其外切核酸酶活性的模型。
