Table_1_Type II Restriction of Bacteriophage DNA With 5hmdU-Derived Base Modifications.docx

To counteract bacterial defense systems, bacteriophages (phages) make extensive base modifications (substitutions) to block endonuclease restriction. Here we evaluated Type II restriction of three thymidine (T or 5-methyldeoxyuridine, 5mdU) modified phage genomes: Pseudomonas phage M6 with 5-(2-aminoethyl)deoxyuridine (5-NedU), Salmonella phage ViI (Vi1) with 5-(2-aminoethoxy)methyldeoxyuridine (5-NeOmdU) and Delftia phage phi W-14 (a.k.a. ΦW-14) with α-putrescinylthymidine (putT). Among >200 commercially available restriction endonucleases (REases) tested, phage M6, ViI, and phi W-14 genomic DNAs (gDNA) show resistance against 48.4, 71.0, and 68.8% of Type II restrictions, respectively. Inspection of the resistant sites indicates the presence of conserved dinucleotide TG or TC (TS, S=C, or G), implicating the specificity of TS sequence as the target that is converted to modified base in the genomes. We also tested a number of DNA methyltransferases (MTases) on these phage DNAs and found some MTases can fully or partially modify the DNA to confer more resistance to cleavage by REases. Phage M6 restriction fragments can be efficiently ligated by T4 DNA ligase. Phi W-14 restriction fragments show apparent reduced rate in E. coli exonuclease III degradation. This work extends previous studies that hypermodified T derived from 5hmdU provides additional resistance to host-encoded restrictions, in parallel to modified cytosines, guanine, and adenine in phage genomes. The results reported here provide a general guidance to use REases to map and clone phage DNA with hypermodified thymidine.

为对抗细菌防御系统，噬菌体（bacteriophages，phages）会通过广泛的碱基修饰（碱基替换）阻断限制性核酸内切酶的酶切作用。本研究针对三种经胸腺嘧啶（T或5-甲基脱氧尿苷，5mdU）修饰的噬菌体基因组的II型限制性酶切特性进行评估：分别为携带5-(2-氨乙基)脱氧尿苷（5-NedU）的假单胞菌噬菌体M6、携带5-(2-氨基乙氧基甲基)脱氧尿苷（5-NeOmdU）的沙门氏菌噬菌体ViI（Vi1），以及携带α-腐胺基胸腺嘧啶（putT）的德尔福特噬菌体phi W-14（又名ΦW-14）。在测试的200余种商用限制性核酸内切酶（restriction endonucleases，REases）中，噬菌体M6、ViI与phi W-14的基因组DNA（genomic DNA，gDNA）分别对48.4%、71.0%及68.8%的II型限制性酶切表现出抗性。对抗性酶切位点的分析显示，其序列中存在保守二核苷酸TG或TC（即TS，其中S代表C或G），这表明TS序列是该类噬菌体基因组中被修饰碱基替换的特异性靶点。本研究同时针对上述噬菌体DNA测试了多种DNA甲基转移酶（DNA methyltransferases，MTases），结果发现部分甲基转移酶可对DNA进行完全或部分修饰，使其进一步获得抵抗限制性内切酶切割的能力。噬菌体M6的限制性酶切片段可通过T4 DNA连接酶实现高效连接。Phi W-14的限制性酶切片段在大肠杆菌外切酶III的降解过程中，表现出显著减慢的降解速率。本研究拓展了此前的相关研究成果：此前研究表明，源自5hmdU的高度修饰胸腺嘧啶，可与噬菌体基因组中经修饰的胞嘧啶、鸟嘌呤及腺嘌呤类似，为噬菌体提供抵抗宿主编码限制性系统的保护能力。本研究所得结果可为利用限制性内切酶定位与克隆携带高度修饰胸腺嘧啶的噬菌体DNA提供通用指导方案。