Fidelity of uracil-initiated base excision DNA repair in DNA polymerase β-proficient and -deficient mouse embryonic fibroblast cell extracts
Fidelity of uracil-initiated base excision DNA repair in DNA polymerase β-proficient and -deficient mouse embryonic fibroblast cell extracts
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DOI：
10.1074/jbc.m106212200
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发表时间：
2001-11-09
影响因子：
4.8
通讯作者：
Mosbaugh, DW
中科院分区：
文献类型：
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作者：
Bennett, SE;Sung, JS;Mosbaugh, DW
使用纯合子小鼠胚胎成纤维细胞DNA聚合酶（+/+）和（-/-）细胞进行尿嘧啶启动的碱基切除DNA修复，以确定与完成修复过程相关的错误频率和突变特异性。在M13mp2 DNA lacZ α基因中的U-A、U-G和U-T靶点上构建了形式I DNA底物。在DNA聚合酶β(+/+)和（-/-）无细胞提取物中均观察到有效的修复。修复在很大程度上依赖于尿嘧啶- dna糖基酶活性，因为添加PBS-2尿嘧啶- dna糖基酶抑制剂（Ugi）蛋白降低了两种类型的无细胞提取物的初始修复率（类似于88%）。在每种情况下，DNA修复补丁的大小主要分布在1到8个核苷酸之间，1个核苷酸修复补丁构成了大约20%的修复事件。将p21肽或蛋白添加到DNA聚合酶β(+/+)无细胞提取物中，短片段（1核苷酸）修复的频率增加了近2倍。当使用DNA聚合酶P（+/+）和（-/-）无细胞提取物时，确定与尿嘧啶-DNA修复相关的碱基取代还原频率为5.7-7.2 x 10（-4）。在这两种情况下，错误频率非常相似，但突变谱明显不同。Ugi的存在与否并没有显著影响错误率或突变特异性。相反，Ugi和p21蛋白的结合促进了与M13mp2 (U-G) DNA修复相关的突变频率的增加。通过前向突变检测产生的突变谱显示，DNA修复合成中的错误主要发生在U-G靶点的位置，并且经常涉及1个碱基的缺失或dTMP的合并。
Uracil-initiated base excision DNA repair was conducted using homozygous mouse embryonic fibroblast DNA polymerase (+/+) and (-/-) cells to determine the error frequency and mutational specificity associated with the completed repair process. Form I DNA substrates were constructed with site-specific uracil residues at U-A, U-G, and U-T targets contained within the lacZ alpha gene of M13mp2 DNA. Efficient repair was observed in both DNA polymerase beta (+/+) and (-/-) cell free extracts. Repair was largely dependent on uracil-DNA glycosylase activity because addition of the PBS-2 uracil-DNA glycosylase inhibitor (Ugi) protein reduced (similar to 88%) the initial rate of repair in both types of cell-free extracts. In each case, the DNA repair patch size was primarily distributed between 1 and 8 nucleotides in length with 1 nucleotide repair patch constituting similar to 20% of the repair events. Addition of p21 peptide or protein to DNA polymerase beta (+/+) cell-free extracts increased the frequency of short-patch (1 nucleotide) repair by similar to2-fold. The base substitution reversion frequency associated with uracil-DNA repair of M13mp2op14 (U-T) DNA was determined to be 5.7-7.2 x 10(-4) when using DNA polymerase P (+/+) and (-/-) cell-free extracts. In these two cases, the error frequency was very similar, but the mutational spectrum was noticeably different. The presence or absence of Ugi did not dramatically influence either the error rate or mutational specificity. In contrast, the combination of Ugi and p21 protein promoted an increase in the mutation frequency associated with repair of M13mp2 (U-G) DNA. Examination of the mutational spectra generated by a forward mutation assay revealed that errors in DNA repair synthesis occurred predominantly at the position of the U-G target and frequently involved a 1-base deletion or incorporation of dTMP.