Takeishi Ayuna, Kogashi Hiroyuki, Odagiri Mizuki, Sasanuma Hiroyuki, Takeda Shunichi, Yasui Manabu, Honma Masamitsu, Suzuki Tetsuya, Kamiya Hiroyuki, Sugasawa Kaoru, Ura Kiyoe, Sassa Akira
Department of Biology, Graduate School of Science, Chiba University, Chiba, Japan.
Department of Radiation Genetics, Graduate School of Medicine, Kyoto University, Yoshida Konoe, Sakyo-ku, Kyoto, Japan.
PLoS One. 2020 Dec 31;15(12):e0244790. doi: 10.1371/journal.pone.0244790. eCollection 2020.
Ribonucleoside triphosphates are often incorporated into genomic DNA during DNA replication. The accumulation of unrepaired ribonucleotides is associated with genomic instability, which is mediated by DNA topoisomerase 1 (Top1) processing of embedded ribonucleotides. The cleavage initiated by Top1 at the site of a ribonucleotide leads to the formation of a Top1-DNA cleavage complex (Top1cc), occasionally resulting in a DNA double-strand break (DSB). In humans, tyrosyl-DNA phosphodiesterases (TDPs) are essential repair enzymes that resolve the trapped Top1cc followed by downstream repair factors. However, there is limited cellular evidence of the involvement of TDPs in the processing of incorporated ribonucleotides in mammals. We assessed the role of TDPs in mutagenesis induced by a single ribonucleotide embedded into DNA. A supF shuttle vector site-specifically containing a single riboguanosine (rG) was introduced into the human lymphoblastoid TK6 cell line and its TDP1-, TDP2-, and TDP1/TDP2-deficient derivatives. TDP1 and TDP2 insufficiency remarkably decreased the mutant frequency caused by an embedded rG. The ratio of large deletion mutations induced by rG was also substantially lower in TDP1/TDP2-deficient cells than wild-type cells. Furthermore, the disruption of TDPs reduced the length of rG-mediated large deletion mutations. The recovery ratio of the propagated plasmid was also increased in TDP1/TDP2-deficient cells after the transfection of the shuttle vector containing rG. The results suggest that TDPs-mediated ribonucleotide processing cascade leads to unfavorable consequences, whereas in the absence of these repair factors, a more error-free processing pathway might function to suppress the ribonucleotide-induced mutagenesis. Furthermore, base substitution mutations at sites outside the position of rG were detected in the supF gene via a TDPs-independent mechanism. Overall, we provide new insights into the mechanism of mutagenesis induced by an embedded ribonucleotide in mammalian cells, which may lead to the fatal phenotype in the ribonucleotide excision repair deficiency.
在DNA复制过程中，核糖核苷三磷酸常常会掺入基因组DNA中。未修复的核糖核苷酸的积累与基因组不稳定相关，这是由DNA拓扑异构酶1（Top1）对嵌入的核糖核苷酸进行加工介导的。Top1在核糖核苷酸位点引发的切割会导致形成Top1-DNA切割复合物（Top1cc），偶尔会导致DNA双链断裂（DSB）。在人类中，酪氨酰-DNA磷酸二酯酶（TDPs）是重要的修复酶，可解决被困的Top1cc，随后由下游修复因子进行处理。然而，在哺乳动物中，关于TDPs参与处理掺入的核糖核苷酸的细胞证据有限。我们评估了TDPs在由嵌入DNA的单个核糖核苷酸诱导的诱变中的作用。将一个特异性含有单个核糖鸟苷（rG）的supF穿梭载体引入人淋巴母细胞TK6细胞系及其TDP1、TDP2和TDP1/TDP2缺陷衍生物中。TDP1和TDP2功能不足显著降低了由嵌入的rG引起的突变频率。在TDP1/TDP2缺陷细胞中，rG诱导的大缺失突变的比例也明显低于野生型细胞。此外，TDPs的缺失缩短了rG介导的大缺失突变的长度。在转染含有rG的穿梭载体后，TDP1/TDP2缺陷细胞中复制质粒的回收率也有所增加。结果表明，TDPs介导的核糖核苷酸加工级联会导致不良后果，而在缺乏这些修复因子的情况下，可能会有一个更无错误的加工途径来抑制核糖核苷酸诱导的诱变。此外，通过一种不依赖TDPs的机制在supF基因中检测到了rG位置以外位点的碱基替换突变。总体而言，我们为哺乳动物细胞中嵌入的核糖核苷酸诱导的诱变机制提供了新的见解，这可能导致核糖核苷酸切除修复缺陷中的致命表型。
