Discipline of Biological Engineering, Indian Institute of Technology Gandhinagar, Gujarat 382355, India.
Cells. 2021 May 29;10(6):1351. doi: 10.3390/cells10061351.
The catalytic domain of most 'cut and paste' DNA transposases have the canonical RNase-H fold, which is also shared by other polynucleotidyl transferases such as the retroviral integrases and the RAG1 subunit of V(D)J recombinase. The RNase-H fold is a mixture of beta sheets and alpha helices with three acidic residues (Asp, Asp, Glu/Asp-DDE/D) that are involved in the metal-mediated cleavage and subsequent integration of DNA. Human THAP9 (hTHAP9), homologous to the well-studied Drosophila P-element transposase (DmTNP), is an active DNA transposase that, although domesticated, still retains the catalytic activity to mobilize transposons. In this study we have modeled the structure of hTHAP9 using the recently available cryo-EM structure of DmTNP as a template to identify an RNase-H like fold along with important acidic residues in its catalytic domain. Site-directed mutagenesis of the predicted catalytic residues followed by screening for DNA excision and integration activity has led to the identification of candidate Ds and Es in the RNaseH fold that may be a part of the catalytic triad in hTHAP9. This study has helped widen our knowledge about the catalytic activity of a functionally uncharacterized transposon-derived gene in the human genome.
大多数“切和粘贴”DNA 转座酶的催化结构域具有典型的 RNase-H 折叠结构，该结构也被其他多核苷酸转移酶共享，如逆转录病毒整合酶和 V(D)J 重组酶的 RAG1 亚基。RNase-H 折叠结构由β片层和α螺旋组成，其中有三个酸性残基（Asp、Asp、Glu/Asp-DDE/D）参与金属介导的 DNA 切割和随后的整合。人类 THAP9（hTHAP9）与研究充分的果蝇 P 元素转座酶（DmTNP）同源，是一种具有活性的 DNA 转座酶，尽管已经驯化，但仍保留了动员转座子的催化活性。在这项研究中，我们使用最近可用的 DmTNP 冷冻电镜结构作为模板，对 hTHAP9 的结构进行建模，以确定其催化结构域中的 RNase-H 样折叠结构以及重要的酸性残基。对预测的催化残基进行定点突变，然后筛选 DNA 切除和整合活性，导致在 RNaseH 折叠中鉴定出候选的 Ds 和 Es，它们可能是 hTHAP9 催化三联体的一部分。这项研究有助于拓宽我们对人类基因组中功能未表征的转座子衍生基因的催化活性的认识。
