Langebio-Cinvestav Sede Irapuato, Km. 9.6 Libramiento Norte Carretera. Irapuato-León, 36821, Irapuato Guanajuato, Mexico.
Laboratorio de Estructura Biomolecular, Centro de Investigación en Alimentación y Desarrollo, A.C. Carretera Gustavo Enrique Astiazarán Rosas Núm. 46, Ejido a La Victoria, 83304, Hermosillo, Sonora, Mexico.
BMC Plant Biol. 2023 Oct 6;23(1):467. doi: 10.1186/s12870-023-04477-4.
The mechanisms and regulation for DNA replication in plant organelles are largely unknown, as few proteins involved in replisome assembly have been biochemically studied. A primase-helicase dubbed Twinkle (T7 gp4-like protein with intramitochondrial nucleoid localization) unwinds double-stranded DNA in metazoan mitochondria and plant organelles. Twinkle in plants is a bifunctional enzyme with an active primase module. This contrast with animal Twinkle in which the primase module is inactive. The organellar primase-helicase of Arabidopsis thaliana (AtTwinkle) harbors a primase module (AtPrimase) that consists of an RNA polymerase domain (RPD) and a Zn + + finger domain (ZFD).
Herein, we investigate the mechanisms by which AtTwinkle recognizes its templating sequence and how primer synthesis and coupling to the organellar DNA polymerases occurs. Biochemical data show that the ZFD of the AtPrimase module is responsible for template recognition, and this recognition is achieved by residues N163, R166, and K168. The role of the ZFD in template recognition was also corroborated by swapping the RPDs of bacteriophage T7 primase and AtPrimase with their respective ZFDs. A chimeric primase harboring the ZFD of T7 primase and the RPD of AtPrimase synthesizes ribonucleotides from the T7 primase recognition sequence and conversely, a chimeric primase harboring the ZFD of AtPrimase and the RPD of T7 primase synthesizes ribonucleotides from the AtPrimase recognition sequence. A chimera harboring the RPDs of bacteriophage T7 and the ZBD of AtTwinkle efficiently synthesizes primers for the plant organellar DNA polymerase.
We conclude that the ZFD is responsible for recognizing a single-stranded sequence and for primer hand-off into the organellar DNA polymerases active site. The primase activity of plant Twinkle is consistent with phylogeny-based reconstructions that concluded that Twinkle´s last eukaryotic common ancestor (LECA) was an enzyme with primase and helicase activities. In plants, the primase domain is active, whereas the primase activity was lost in metazoans. Our data supports the notion that AtTwinkle synthesizes primers at the lagging-strand of the organellar replication fork.
植物细胞器中 DNA 复制的机制和调控在很大程度上是未知的，因为很少有参与复制体组装的蛋白质被生物化学研究过。一种被称为 Twinkle（具有线粒体核定位的 T7 gp4 样蛋白）的引发酶-解旋酶在后生动物线粒体和植物细胞器中解开双链 DNA。植物中的 Twinkle 是一种具有活性引发酶模块的双功能酶。这与动物 Twinkle 形成对比，动物 Twinkle 的引发酶模块没有活性。拟南芥的细胞器引发酶-解旋酶（AtTwinkle）含有一个引发酶模块（AtPrimase），该模块由 RNA 聚合酶结构域（RPD）和 Zn++ 手指结构域（ZFD）组成。
本文研究了 AtTwinkle 识别其模板序列的机制，以及引物合成和与细胞器 DNA 聚合酶偶联的机制。生化数据表明，AtPrimase 模块的 ZFD 负责模板识别，这种识别是通过残基 N163、R166 和 K168 实现的。ZFD 在模板识别中的作用也通过用噬菌体 T7 引发酶和 AtPrimase 的 RPD 与其各自的 ZFD 替换来得到证实。一种含有 T7 引发酶 ZFD 和 AtPrimase RPD 的嵌合引发酶可以从 T7 引发酶识别序列合成核糖核苷酸，相反，一种含有 AtPrimase ZFD 和 T7 引发酶 RPD 的嵌合引发酶可以从 AtPrimase 识别序列合成核糖核苷酸。一种含有噬菌体 T7 的 RPD 和 AtTwinkle 的 ZBD 的嵌合体有效地为植物细胞器 DNA 聚合酶合成引物。
我们得出结论，ZFD 负责识别单链序列，并将引物传递到细胞器 DNA 聚合酶的活性位点。植物 Twinkle 的引发酶活性与基于系统发育的重建一致，该重建得出结论，Twinkle 的最后一个真核共同祖先（LECA）是一种具有引发酶和解旋酶活性的酶。在植物中，引发酶结构域是活跃的，而在后生动物中，引发酶活性丧失。我们的数据支持 AtTwinkle 在细胞器复制叉的滞后链上合成引物的观点。
