Feng Xu, Zhang Baochang, Xu Ruyi, Gao Zhe, Liu Xiaotong, Yuan Guanhua, Ishino Sonoko, Feng Mingxia, Shen Yulong, Ishino Yoshizumi, She Qunxin
CRISPR and Archaea Biology Research Center, Microbial Technology Institute and State Key Laboratory of Microbial Technology, Shandong University, Qingdao, China.
Department of Bioscience and Biotechnology, Graduate School of Bioresource and Bioenvironmental Sciences, Kyushu University, Fukuoka, Japan.
Front Microbiol. 2021 Dec 20;12:802670. doi: 10.3389/fmicb.2021.802670. eCollection 2021.
Abasic sites are among the most abundant DNA lesions encountered by cells. Their replication requires actions of specialized DNA polymerases. Herein, two archaeal specialized DNA polymerases were examined for their capability to perform translesion DNA synthesis (TLS) on the lesion, including Dpo2 of B-family, and Dpo4 of Y-family. We found neither Dpo2 nor Dpo4 is efficient to complete abasic sites bypass alone, but their sequential actions promote lesion bypass. Enzyme kinetics studies further revealed that the Dpo4's activity is significantly inhibited at +1 to +3 site past the lesion, at which Dpo2 efficiently extends the primer termini. Furthermore, their activities are inhibited upon synthesis of 5-6 nt TLS patches. Once handed over to Dpo1, these substrates basically inactivate its exonuclease, enabling the transition from proofreading to polymerization of the replicase. Collectively, by functioning as an "extender" to catalyze further DNA synthesis past the lesion, Dpo2 bridges the activity gap between Dpo4 and Dpo1 in the archaeal TLS process, thus achieving more efficient lesion bypass.
无碱基位点是细胞所遇到的最为常见的DNA损伤之一。它们的复制需要特殊DNA聚合酶的作用。在此，我们检测了两种古细菌特殊DNA聚合酶在该损伤上进行跨损伤DNA合成（TLS）的能力，包括B家族的Dpo2和Y家族的Dpo4。我们发现，单独的Dpo2和Dpo4都不能有效地完成无碱基位点的跨越，但它们的顺序作用可促进损伤跨越。酶动力学研究进一步揭示，在损伤位点下游的+1至+3位点，Dpo4的活性受到显著抑制，而Dpo2能在此有效地延伸引物末端。此外，在合成5 - 6个核苷酸的TLS片段后，它们的活性受到抑制。一旦将这些底物交给Dpo1，基本上会使其核酸外切酶失活，从而实现从复制酶的校对功能向聚合功能的转变。总的来说，通过作为“延伸酶”催化损伤位点下游的进一步DNA合成，Dpo2在古细菌TLS过程中弥合了Dpo4和Dpo1之间的活性差距，从而实现了更有效的损伤跨越。
