Cloning of cDNA encoding Drosophila poly(ADP-ribose) polymerase: leucine zipper in the auto-modification domain.

We have isolated cDNA clones for a Drosophila poly(ADP-ribose) polymerase (PARP; EC 2.4.2.30) by screening a lambda gt11 cDNA library with a Drosophila partial cDNA fragment. The Drosophila PARP probe was obtained by the polymerase chain reaction with heterologous primers deduced from conserved amino acids in the mammalian, chicken, amphibian, and fish sequences. The Drosophila PARP mRNA is 3.2 kb in length and is expressed in the early stages of development. The PARP protein of 994 amino acids contains two zinc-finger motifs and an NAD-binding motif, which are conserved among different species. Interestingly, the heptad leucine repeat in an alpha-helix was found in Drosophila PARP. Alignments of the auto-modification domains of various species showed the repeated hydrophobic amino acids on the same face of the helix that make the coiled-coil configuration in the mammalian and chicken sequences. The presence of a leucine-zipper motif in the auto-modification domain suggests that this motif might be responsible for protein-protein interaction between PARP and physiological acceptors. PARP may have novel functions, possibly involving its homo- and/or heterodimerization with other nuclear leucine-zipper proteins and its regulation by ADP-ribosylation. IMAGES:

本研究通过以果蝇部分cDNA片段作为探针，筛选λgt11 cDNA文库，成功分离得到果蝇聚(ADP-核糖)聚合酶（poly(ADP-ribose) polymerase，PARP；EC 2.4.2.30）的cDNA克隆。本研究所用的果蝇PARP探针，是以哺乳类、鸡、两栖动物及鱼类序列中的保守氨基酸区域推导得到的异源引物，通过聚合酶链式反应（polymerase chain reaction）扩增获得。果蝇PARP的mRNA长度为3.2 kb，在发育早期阶段即有表达。该PARP蛋白由994个氨基酸残基构成，包含两个锌指基序（zinc-finger motif）与一个NAD结合基序（NAD-binding motif），这些结构域在不同物种间均具有保守性。值得注意的是，本研究在果蝇PARP中发现了α螺旋内的亮氨酸七肽重复序列。对不同物种的自身修饰结构域进行序列比对后发现，在哺乳类与鸡类的序列中，螺旋同一侧存在重复的疏水氨基酸残基，该结构可形成卷曲螺旋构型。自身修饰结构域中存在亮氨酸拉链基序，提示该基序可能介导PARP与其生理作用受体之间的蛋白质-蛋白质相互作用。PARP可能具备全新的生物学功能，其机制可能涉及与其他核内亮氨酸拉链蛋白形成同源/异源二聚体，以及通过ADP核糖基化修饰实现调控。图像：