MECHANISTIC STUDIES OF IONIZING-RADIATION AND OXIDATIVE MUTAGENESIS - GENETIC-EFFECTS OF A SINGLE 8-HYDROXYGUANINE (7-HYDRO-8-OXOGUANINE) RESIDUE INSERTED AT A UNIQUE SITE IN A VIRAL GENOME
MECHANISTIC STUDIES OF IONIZING-RADIATION AND OXIDATIVE MUTAGENESIS - GENETIC-EFFECTS OF A SINGLE 8-HYDROXYGUANINE (7-HYDRO-8-OXOGUANINE) RESIDUE INSERTED AT A UNIQUE SITE IN A VIRAL GENOME
复制标题
DOI：
10.1021/bi00482a011
复制
发表时间：
1990-07-31
期刊：
影响因子：
2.9
通讯作者：
ESSIGMANN, JM
中科院分区：
文献类型：
--
作者：
WOOD, ML;DIZDAROGLU, M;ESSIGMANN, JM
用T4 RNA连接酶构建了一个含有8-羟基鸟嘌呤（7-hydro-8-oxoguanine; G8-OH）残基的脱氧五核苷酸。通过将化学合成的受体分子d（GCTA）连接到加合的供体8-羟基-2“-脱氧鸟苷5”，3“-二磷酸来制备五聚体d（GCTAG 8-OH）p。受体被有效地转化为反应产物（>95%），并且最终产物产率为50%。在3“-脱磷酸化之后，通过UV光谱、高压液相色谱和酶水解释放的核苷的气相色谱-质谱表征五聚体。d（GCTAG 8-OH）和未修饰的对照均通过使用[γ-氨基]磷酸化。32 P]ATP，并通过DNA连接酶共价结合到M13 mp 19衍生物的另外的双链体基因组中的独特NheI限制性位点处的五个碱基缺口中。连接产物在噬菌体lacZ α的读框内琥珀密码子（5“-TAG-3”）（基因组位置6276）的3“残基处含有G8-OH。基因该加合物是独特限制性位点中无义密码子的一部分，以便于鉴定和选择由大肠杆菌中修饰的基因组复制产生的突变体。对照和加合物五聚体都以最大理论效率的50%连接到基因组中，并且几乎所有（约90%）位点特异性加合物产物都具有在5“和3”末端共价连接的五核苷酸。在NheI位点的G8-OH损伤抑制了200倍过量的NheI对该位点的裂解。转化大肠具有独特修饰的单链基因组的大肠杆菌菌株DL 7产生了0.5-1.0%的子代噬菌体显示G →在G8-OH的原始位置处的T颠换突变;在含有鸟嘌呤代替G8-OH的对照基因组中未观察到此类突变。含有G8-OH的载体也转化50-90%的未修饰的对照有效，表明加合物可以是弱细胞毒性和致突变的噬菌体基因组。
T4 RNA ligase was used to construct a deoxypentanucleotide containing a single 8-hydroxyguanine (7-hydro-8-oxoguanine; G8-OH) residue, which is one of the putatively mutagenic DNA adducts produced by oxidants and ionizing radiation. The pentamer d(GCTAG8-OH)p was prepared by the ligation of a chemically synthesized acceptor molecule, d(GCTA), to an adducted donor, 8-hydroxy-2''-deoxyguanosine 5'',3''-bisphosphate. The acceptor was efficiently converted to the reaction product (>95%), and the final product yield was 50%. Following 3''-dephosphorylation, the pentamer was characterized by UV spectroscopy, by high-pressure liquid chromatography, and by gas chromatography-mass spectrometry of the nucleosides released by enzymatic hydrolysis. Both d(GCTAG8-OH) and an unmodified control were 5''-phosphorylated by using [.gamma.-32P]ATP and incorporated covalently by DNA ligase into a five-base gap at a unique NheI restriction site in the otherwise duplex genome of an M13mp19 derivative. The ligation product contained G8-OH at the 3'' residue of an in-frame amber codon (5''-TAG-3'') (genome position 6276) of the phage lacZ.alpha. gene. The adduct was part of a nonsense codon in a unique restriction site in order to facilitate the identification and selection of mutants generated by the replication of the modified genome in Escherichia coli. Both control and adducted pentamers ligated into the genome at 50% of the maximum theoretical efficiency, and nearly all (.apprx.90%) of the site-specifically adducted products possessed pentanucleotides that were covalently linked at both 5'' and 3'' termini. The G8-OH lesion in the NheI site inhibited the cleavage of the site by a 200-fold excess of NheI. Transformation of E. coli strain DL7 with the uniquely modified single-stranded genome resulted in .apprx. 0.5-1.0% of the progeny phage showing the G .fwdarw. T transversion mutation at the original position of G8-OH; no such mutations were observed from control genomes containing guanine in place of G8-OH. The vector containing G8-OH also transformed 50-90% as efficiently as the unmodified control, indicating that the adduct can be both weakly cytotoxic and mutagenic to the phage genome.