Liu Yuan, Zhang Haihua, Veeraraghavan Janaki, Bambara Robert A, Freudenreich Catherine H
Department of Biochemistry and Biophysics, University of Rochester School of Medicine and Dentistry, Rochester, New York 14642,USA.
Mol Cell Biol. 2004 May;24(9):4049-64. doi: 10.1128/MCB.24.9.4049-4064.2004.
Flap endonuclease 1 (FEN1) is a central component of Okazaki fragment maturation in eukaryotes. Genetic analysis of Saccharomyces cerevisiae FEN1 (RAD27) also reveals its important role in preventing trinucleotide repeat (TNR) expansion. In humans such expansion is associated with neurodegenerative diseases. In vitro, FEN1 can inhibit TNR expansion by employing its endonuclease activity to compete with DNA ligase I. Here we employed two yeast FEN1 nuclease mutants, rad27-G67S and rad27-G240D, to further define the mechanism by which FEN1 prevents TNR expansion. Using a yeast artificial chromosome system that can detect both TNR instability and fragility, we demonstrate that the G240D but not the G67S mutation increases both the expansion and fragility of a CTG tract in vivo. In vitro, the G240D nuclease is proficient in cleaving a fixed nonrepeat double flap; however, it exhibits severely impaired cleavage of both nonrepeat and CTG-containing equilibrating flaps. In contrast, wild-type FEN1 and the G67S mutant exhibit more efficient cleavage on an equilibrating flap than on a fixed CTG flap. The degree of TNR expansion and the amount of chromosome fragility observed in the mutant strains correlate with the severity of defective flap cleavage in vitro. We present a model to explain how flap equilibration and the unique tracking mechanism of FEN1 can collaborate to remove TNR flaps and prevent repeat expansion.
瓣内切核酸酶1（FEN1）是真核生物中冈崎片段成熟过程的核心组成部分。对酿酒酵母FEN1（RAD27）的遗传分析也揭示了其在防止三核苷酸重复序列（TNR）扩增方面的重要作用。在人类中，这种扩增与神经退行性疾病相关。在体外，FEN1可通过利用其内切核酸酶活性与DNA连接酶I竞争来抑制TNR扩增。在此，我们使用了两种酵母FEN1核酸酶突变体rad27 - G67S和rad27 - G240D，以进一步明确FEN1防止TNR扩增的机制。利用一种能够检测TNR不稳定性和脆性的酵母人工染色体系统，我们证明G240D突变而非G67S突变在体内增加了CTG序列的扩增和脆性。在体外，G240D核酸酶能够高效切割固定的非重复双瓣结构；然而，它对非重复和含CTG的平衡瓣的切割能力严重受损。相比之下，野生型FEN1和G67S突变体在平衡瓣上的切割效率高于固定的CTG瓣。在突变菌株中观察到的TNR扩增程度和染色体脆性与体外瓣切割缺陷的严重程度相关。我们提出了一个模型来解释瓣平衡和FEN1独特的追踪机制如何协同作用以去除TNR瓣并防止重复序列扩增。
