Sanderson R J, Mosbaugh D W
Departments of Agricultural Chemistry and Biochemistry and Biophysics, Oregon State University, Corvallis, Oregon 97331, USA.
J Biol Chem. 1998 Sep 18;273(38):24822-31. doi: 10.1074/jbc.273.38.24822.
The fidelity of DNA synthesis associated with uracil-initiated base excision repair was measured in human whole cell extracts. An M13mp2 lacZalpha DNA-based reversion assay was developed to assess the error frequency of DNA repair synthesis at a site-specific uracil residue. All three possible base substitution errors were detected at the uracil target causing reversion of opal codon 14 in the Escherichia coli lacZalpha gene. Using human glioblastoma U251 whole cell extracts, approximately 50% of the heteroduplex uracil-containing DNA substrate was completely repaired, as determined by the insensitivity of form I DNA reaction products to cleavage by a combined treatment of E. coli uracil-DNA glycosylase and endonuclease IV. The majority of repair occurred by the uracil-initiated base excision repair pathway, since the addition of the bacteriophage PBS2 uracil-DNA glycosylase inhibitor protein to extracts significantly blocked this process. In addition, the formation of repaired form I DNA molecules occurred concurrently with limited DNA synthesis, which was largely restricted to the HinfI DNA fragment initially containing the uracil residue and specific to the uracil-containing DNA strand. Based on the reversion frequency of repaired M13mp2 DNA, the fidelity of DNA repair synthesis at the target was determined to be about one misincorporated nucleotide per 1900 repaired uracil residues. The major class of base substitutions propagated transversion mutations, which were distributed almost equally between T to G and T to A changes in the template. A similar mutation frequency was also observed using whole cell extracts from human colon adenocarcinoma LoVo cells, suggesting that mismatch repair did not interfere with the fidelity measurements.
在人全细胞提取物中测量了与尿嘧啶引发的碱基切除修复相关的DNA合成保真度。开发了一种基于M13mp2 lacZα DNA的回复突变测定法，以评估在特定位置的尿嘧啶残基处DNA修复合成的错误频率。在尿嘧啶靶位点检测到所有三种可能的碱基替代错误，导致大肠杆菌lacZα基因中的乳白密码子14回复突变。使用人胶质母细胞瘤U251全细胞提取物，约50%的含异源双链尿嘧啶的DNA底物被完全修复，这是通过I型DNA反应产物对大肠杆菌尿嘧啶-DNA糖基化酶和核酸内切酶IV联合处理的切割不敏感来确定的。大多数修复是通过尿嘧啶引发的碱基切除修复途径发生的，因为向提取物中添加噬菌体PBS2尿嘧啶-DNA糖基化酶抑制蛋白会显著阻断这一过程。此外，修复后的I型DNA分子的形成与有限的DNA合成同时发生，这在很大程度上仅限于最初含有尿嘧啶残基的HinfI DNA片段，并且特定于含尿嘧啶的DNA链。根据修复后的M13mp2 DNA的回复突变频率，确定靶位点DNA修复合成的保真度约为每1900个修复的尿嘧啶残基中有一个错配掺入的核苷酸。主要的碱基替代类别传播了颠换突变，这些突变在模板中的T到G和T到A变化之间几乎均匀分布。使用人结肠腺癌LoVo细胞的全细胞提取物也观察到了类似的突变频率，这表明错配修复不会干扰保真度测量。
