Development and use of an in vitro HSV-tk forward mutation assay to study eukaryotic DNA polymerase processing of DNA alkyl lesions
Development and use of an in vitro HSV-tk forward mutation assay to study eukaryotic DNA polymerase processing of DNA alkyl lesions
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DOI：
10.1093/nar/25.7.1450
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发表时间：
1997-04-01
影响因子：
14.9
通讯作者：
Vargo, PL
中科院分区：
文献类型：
--
作者：
Eckert, KA;Hile, SE;Vargo, PL
我们开发了一种体外 DNA 聚合酶正向突变测定法，使用含有单纯疱疹病毒 1 型胸苷激酶 (HSV-tk) 基因的受损 DNA 模板，该定量方法使用互补链杂交到有缺口的双链 DNA 分子和氯霉素选择，该设计确保对体外过程中产生的 DNA 链衍生的突变进行独家分析 合成。我们检查了小牛胸腺聚合酶 α-引物酶、聚合酶 β 和核酸外切酶缺陷型 Klenow 聚合酶催化的 DNA 合成的准确性，使用未修饰的 DNA 模板，聚合酶 β 对 HSV-tk 基因座内二核苷酸重复序列中两个碱基的丢失显示出独特的特异性，用以下方法处理 DNA 模板 N-乙基-N-亚硝基脲导致 DNA 合成的剂量依赖性抑制，同时突变频率增加。对于所检查的三种聚合酶，测量了类似的剂量反应曲线；因此，DNA聚合酶的特性似乎不会影响乙基损伤的诱变能力。 HSV-fk 系统的独特之处在于，可以对人类细胞、细菌细胞和体外 DNA 合成反应中单个靶序列的损伤诱导突变进行定量和定性分析。
We have developed an in vitro DNA polymerase forward mutation assay using damaged DNA templates that contain the herpes simplex virus type 1 thymidine kinase (HSV-tk) gene, The quantitative method uses complementary strand hybridization to gapped duplex DNA molecules and chloramphenicol selection, This design ensures exclusive analysis of mutations derived from the DNA strand produced during in vitro synthesis. We have examined the accuracy of DNA synthesis catalyzed by calf thymus polymerase alpha-primase, polymerase beta and exonuclease-deficient Klenow polymerase, Using unmodified DNA templates, polymerase beta displays a unique specificity for the loss of two bases in a dinucleotide repeat sequence within the HSV-tk locus, Treatment of the DNA template with N-ethyl-N-nitrosourea resulted in a dose-dependent inhibition of DNA synthesis concomitant with an increased mutation frequency. Similar dose-response curves were measured for the three polymerases examined; thus the identity of the DNA polymerase does not appear to affect the mutagenic potency of ethyl lesions. The HSV-fk system is unique in that damage-induced mutagenesis can be analyzed both quantitatively and qualitatively in human cells, in bacterial cells and in in vitro DNA synthesis reactions at a single target sequence.