Stimulation of eukaryotic flap endonuclease-1 activities by proliferating cell nuclear antigen (PCNA) is independent of its in vitro interaction via a consensus PCNA binding region
Stimulation of eukaryotic flap endonuclease-1 activities by proliferating cell nuclear antigen (PCNA) is independent of its in vitro interaction via a consensus PCNA binding region
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DOI：
10.1074/jbc.m103397200
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发表时间：
2001-09-28
影响因子：
4.8
通讯作者：
Shen, BH
中科院分区：
文献类型：
--
作者：
Frank, G;Qiu, JH;Shen, BH
人Rap核酸内切酶-1（hFEN-1）和增殖细胞核抗原（PCNA）之间的相互作用代表了参与DNA代谢途径的多个功能蛋白之间相互作用的良好模型。在人FEN-1（hFEN-1）的C末端的9个保守氨基酸残基（残基Gln-337至Lys-345）的区域被证明负责与PCNA的相互作用。我们目前的研究表明hFEN-1中的4个氨基酸残基（Leu-340，Asp-341，Phe-343和Phe-344）是人PCNA（hPCNA）相互作用的关键。在来自各种物种的各种蛋白质中，一个保守的PCNA相互作用基序已被定义为Q（1）X（2）X（3）（L/I）（4）X5 X6 F7（F/Y）（8），尽管我们的结果未能暗示Q（1）（hFEN-1中的Gln-337）是一个关键残基。令人惊讶的是，所有hFEN-1突变体，包括L340 A、D341 A、F343 A和F344 A，都保留了hPCNA介导的外切和瓣状核酸内切酶活性的刺激。此外，我们的体外试验表明hPCNA未能与scRad 27（FEN-1的酵母同源物）核酸酶结合。然而，它的核酸酶活性显着增强的hPCNA的存在。构建了另外四个酿酒酵母scRad 27突变体，包括多个丙氨酸突变体和整个PCNA结合区的缺失突变体，以证实这一结果。所有这些突变体都保留了PCNA驱动的核酸酶活性刺激。因此，我们得出结论，刺激真核细胞hFEN-1核酸酶活性的PCNA是独立的，其在体外的相互作用，通过PCNA结合区。
Interaction between human Rap endonuclease-1 (hFEN-1) and proliferating cell nuclear antigen (PCNA) represents a good model for interactions between multiple functional proteins involved in DNA metabolic pathways. A region of 9 conserved amino acid residues (residues Gln-337 through Lys-345) in the C terminus of human FEN-1 (hFEN-1) was shown to be responsible for the interaction with PCNA. Our current study indicates that 4 amino acid residues in hFEN-1 (Leu-340, Asp-341, Phe-343, and Phe-344) are critical for human PCNA (hPCNA) interaction. A conserved PCNA interaction motif in various proteins from assorted species has been defined as Q(1)X(2)X(3)(L/I)(4)X5X6F7(F/Y)(8), although our results fail to implicate Q(1) (Gln-337 in hFEN-1) as a crucial residue. Surprisingly, all hFEN-1 mutants, including L340A, D341A, F343A, and F344A, retained hPCNA-mediated stimulation of both exo- and flap endonuclease activities. Furthermore, our in vitro assay showed that hPCNA failed to bind to the scRad27 (yeast homolog of FEN-1) nuclease. However, its nuclease activities were significantly enhanced in the presence of hPCNA. Four additional Saccharomyces cerevisiae scRad27 mutants, including multiple alanine mutants and a deletion mutant of the entire PCNA binding region, were constructed to confirm this result. All of these mutants retained PCNA-driven nuclease activity stimulation. We therefore conclude that stimulation of eukaryotic hFEN-1 nuclease activities by PCNA is independent of its in vitro interaction via the PCNA binding region.