Guérineau Marc, Bessa Luiza, Moriau Séverine, Lescop Ewen, Bontems François, Mathy Nathalie, Guittet Eric, Bischerour Julien, Bétermier Mireille, Morellet Nelly
Université Paris-Saclay, CEA, CNRS, Institute for Integrative Biology of the Cell (I2BC), 1 Avenue de la Terrasse, 91198, Gif sur Yvette Cedex, France.
Université Paris-Saclay, CNRS, Institut de Chimie des Substances Naturelles, UPR 2301, 1 Avenue de la Terrasse, 91198, Gif sur Yvette Cedex, France.
Mob DNA. 2021 Apr 29;12(1):12. doi: 10.1186/s13100-021-00240-4.
Transposons are mobile genetic elements that colonize genomes and drive their plasticity in all organisms. DNA transposon-encoded transposases bind to the ends of their cognate transposons and catalyze their movement. In some cases, exaptation of transposon genes has allowed novel cellular functions to emerge. The PiggyMac (Pgm) endonuclease of the ciliate Paramecium tetraurelia is a domesticated transposase from the PiggyBac family. It carries a core catalytic domain typical of PiggyBac-related transposases and a short cysteine-rich domain (CRD), flanked by N- and C-terminal extensions. During sexual processes Pgm catalyzes programmed genome rearrangements (PGR) that eliminate ~ 30% of germline DNA from the somatic genome at each generation. How Pgm recognizes its DNA cleavage sites in chromatin is unclear and the structure-function relationships of its different domains have remained elusive.
We provide insight into Pgm structure by determining the fold adopted by its CRD, an essential domain required for PGR. Using Nuclear Magnetic Resonance, we show that the Pgm CRD binds two Zn ions and forms an unusual binuclear cross-brace zinc finger, with a circularly permutated treble-clef fold flanked by two flexible arms. The Pgm CRD structure clearly differs from that of several other PiggyBac-related transposases, among which is the well-studied PB transposase from Trichoplusia ni. Instead, the arrangement of cysteines and histidines in the primary sequence of the Pgm CRD resembles that of active transposases from piggyBac-like elements found in other species and of human PiggyBac-derived domesticated transposases. We show that, unlike the PB CRD, the Pgm CRD does not bind DNA. Instead, it interacts weakly with the N-terminus of histone H3, whatever its lysine methylation state.
The present study points to the structural diversity of the CRD among transposases from the PiggyBac family and their domesticated derivatives, and highlights the diverse interactions this domain may establish with chromatin, from sequence-specific DNA binding to contacts with histone tails. Our data suggest that the Pgm CRD fold, whose unusual arrangement of cysteines and histidines is found in all PiggyBac-related domesticated transposases from Paramecium and Tetrahymena, was already present in the ancestral active transposase that gave rise to ciliate domesticated proteins.
转座子是可移动的遗传元件，可在所有生物体的基因组中定殖并推动其可塑性。DNA转座子编码的转座酶与其同源转座子的末端结合并催化其移动。在某些情况下，转座子基因的功能扩展使得新的细胞功能得以出现。纤毛虫四膜虫的PiggyMac（Pgm）内切核酸酶是一种源自PiggyBac家族的驯化转座酶。它具有PiggyBac相关转座酶典型的核心催化结构域和一个短的富含半胱氨酸的结构域（CRD），两侧是N端和C端延伸。在有性生殖过程中，Pgm催化程序性基因组重排（PGR），在每一代中从体细胞基因组中消除约30%的生殖系DNA。Pgm如何识别其在染色质中的DNA切割位点尚不清楚，其不同结构域的结构-功能关系也仍然难以捉摸。
我们通过确定其CRD（PGR所需的必需结构域）所采用的折叠来深入了解Pgm的结构。利用核磁共振，我们表明Pgm CRD结合两个锌离子并形成一个不寻常的双核交叉支撑锌指，其具有一个环形排列的高音谱号折叠，两侧是两个柔性臂。Pgm CRD的结构明显不同于其他几种PiggyBac相关转座酶，其中包括已被充分研究的粉纹夜蛾的PB转座酶。相反，Pgm CRD一级序列中半胱氨酸和组氨酸的排列类似于在其他物种中发现的piggyBac样元件的活性转座酶以及人类源自PiggyBac的驯化转座酶。我们表明，与PB CRD不同，Pgm CRD不结合DNA。相反，无论其赖氨酸甲基化状态如何，它都与组蛋白H3的N端弱相互作用。
本研究指出了PiggyBac家族转座酶及其驯化衍生物中CRD的结构多样性，并强调了该结构域可能与染色质建立的多种相互作用，从序列特异性DNA结合到与组蛋白尾巴的接触。我们的数据表明，在四膜虫和草履虫所有源自PiggyBac相关的驯化转座酶中发现的Pgm CRD折叠中半胱氨酸和组氨酸的不寻常排列，已经存在于产生纤毛虫驯化蛋白的祖先活性转座酶中。
