Schreiber V, Molinete M, Boeuf H, de Murcia G, Ménissier-de Murcia J
Institut de Biologie Moléculaire et Cellulaire, Centre National de la Recherche Scientifique, Strasbourg, France.
EMBO J. 1992 Sep;11(9):3263-9. doi: 10.1002/j.1460-2075.1992.tb05404.x.
Poly(ADP-ribose) polymerase (PARP, EC 2.4.2.30) is a zinc finger DNA-binding protein involved in DNA repair processes in eukaryotes. By deletion and extensive site-directed mutagenesis, its DNA-binding domain fused to the N-terminus of beta-galactosidase was shown to contain a nuclear localization signal (NLS) of the form KRK-X(11)-KKKSKK (residues 207-226). In vitro, both the DNA-binding capacity and the polymerizing activity of PARP are independent of the nuclear location function. Each basic cluster is essential but not sufficient on its own for this function, while both motifs together are. Crucial basic amino acids (K207, R208 and K222) in each of these two motifs are required for nuclear homing. The results presented here support the concept that the human PARP NLS is an autonomous functional element and belongs to the class of bipartite NLSs. We show that the linear distance between the two basic clusters is not crucial. Insertional mutation analysis leading to a partial reversion of the cytoplasmic phenotype displayed by the mutant K222I highlights the crucial positioning of this lysine. The structure-function relationship of the second cluster of basic residues is discussed.
聚（ADP - 核糖）聚合酶（PARP，EC 2.4.2.30）是一种锌指DNA结合蛋白，参与真核生物的DNA修复过程。通过缺失和广泛的定点诱变，发现其与β - 半乳糖苷酶N端融合的DNA结合结构域含有形式为KRK - X(11) - KKKSKK（残基207 - 226）的核定位信号（NLS）。在体外，PARP的DNA结合能力和聚合活性均与核定位功能无关。每个碱性簇对于该功能而言都是必不可少的，但单独存在时并不充分，而两个基序共同存在时则是充分的。这两个基序中的每一个中的关键碱性氨基酸（K207、R208和K222）对于核归巢都是必需的。本文给出的结果支持这样一种概念，即人PARP NLS是一个自主功能元件，属于双分型NLSs类别。我们表明两个碱性簇之间的线性距离并不关键。导致突变体K222I所显示的细胞质表型部分逆转的插入突变分析突出了该赖氨酸的关键定位。讨论了第二组碱性残基的结构 - 功能关系。
