Characterization of argonaute nucleases from mesophilic bacteria Pseudobutyrivibrio ruminis  

作　　者：Xiaoyi Xu Hao Yang Huarong Dong Xiao Li Qian Liu Yan Feng 

机构地区：[1]State Key Laboratory of Microbial Metabolism,School of Life Sciences and Biotechnology,Shanghai Jiao Tong University,Shanghai 200240,People’s Republic of China

出　　处：《Bioresources and Bioprocessing》2024年第1期1244-1256,共13页生物资源与生物加工(英文)

基　　金：supported by the Ministry of Science and Technology(2020YFA0907700);National Natural Science Foundation of China(31770078,32270051);Shanghai Pilot Program for Basic Research-Shanghai Jiao Tong University(21TQ1400204).

摘　　要：Mesophilic Argonautes(Agos)from microbial resources have received significant attention due to their potential applications in genome editing and molecular diagnostics.This study characterizes a novel Ago from Pseudobutyrivi-brio ruminis(PrAgo),which can cleave single-stranded DNA using guide DNA(gDNA).PrAgo,functioning as a multi-turnover enzyme,effectively cleaves DNA using 5′-phosphate gDNA,14-30 nucleotides in length,in the presence of both Mn2+and Mg2+ions.PrAgo demonstrates DNA cleavage activity over a broad pH range(pH 4-12),with optimal activity at pH 11.As a mesophilic enzyme,PrAgo cleaves efficiently DNA at temperatures ranging from 25 to 65°C,particularly at 65°C.PrAgo does not show strong preferences for the 5′-nucleotide in gDNA.It shows high tolerance for single-base mismatches,except at positions 13 and 15 of gDNA.Continuous double-nucleotide mismatches at positions 10-16 of gDNA significantly reduce cleavage activity.Furthermore,PrAgo mediates DNA-guided DNA cleavage of AT-rich double stranded DNA at 65°C.Additionally,molecular dynamic simulations suggest that interactions between the PAZ domain and different nucleic acids strongly influence cleavage efficiency.These findings expand our understanding of Protokaryotic Agos and their potential applications in biotechnology.

关 键 词：Pseudobutyrivibrio ruminis Argonaute Mesophilic Argonaute Prokaryotic Argonaute ENDONUCLEASE DNA cleavage 

分 类 号：O62[理学—有机化学]