Eckert K A, Hile S E
The Jake Gittlen Cancer Research Institute, The Pennsylvania State University College of Medicine, PO Box 850, Hershey, PA 17033, USA.
Mutat Res. 1998 Dec 3;422(2):255-69. doi: 10.1016/s0027-5107(98)00206-1.
We have analyzed the mutational spectra produced during in vitro DNA synthesis by DNA polymerase alpha-primase and DNA polymerase beta. The polymerase mutation frequency as measured in the in vitro herpes simplex virus thymidine kinase (HSV-tk) forward assay was increased when reactions utilized single-stranded DNA templates randomly modified by 20 mM N-ethyl-N-nitrosourea (ENU), relative to solvent-treated templates. A 20- to 50-fold increase in the frequency of G-->A transition mutations was observed for both polymerases, as expected due to mispairing by O6-ethylguanine lesions. Strikingly, ENU treatment of the template also resulted in a five- to 12-fold increased frequency of frameshift errors at heteropolymeric (non-repetitive) template sequences produced by polymerase beta and polymerase alpha-primase, respectively. The increased proportion of frameshift mutations at heteropolymeric sequences relative to homopolymeric (repetitive) sequences produced by each polymerase in response to ENU damage was statistically significant. For polymerase alpha-primase, one-base deletion errors at template guanine residues was the second most frequent mutational event, observed at a frequency only four-fold lower than the G-->A transition frequency. In the polymerase beta reactions, the frequency of insertion errors at homopolymeric (repetitive) sequences was increased six-fold using alkylated templates, relative to solvent controls. The frequency of such insertion errors was only three-fold lower than the frequency of G-->A transition errors by polymerase beta. Although ENU is generally regarded as a potent base substitution mutagen, these data show that monofunctional alkylating agents are capable of inducing frameshift mutations in vitro. Alkylation-induced frameshift mutations occur in both repetitive and non-repetitive DNA sequences; however, the mutational specificity is dependent upon the DNA polymerase.
我们分析了DNA聚合酶α-引发酶和DNA聚合酶β在体外DNA合成过程中产生的突变谱。在体外单纯疱疹病毒胸苷激酶（HSV-tk）正向试验中测得的聚合酶突变频率，当反应使用经20 mM N-乙基-N-亚硝基脲（ENU）随机修饰的单链DNA模板时，相对于溶剂处理的模板有所增加。正如预期的那样，由于O6-乙基鸟嘌呤损伤导致错配，两种聚合酶的G→A转换突变频率均增加了20至50倍。引人注目的是，ENU处理模板还分别导致由聚合酶β和聚合酶α-引发酶产生的异聚体（非重复）模板序列处的移码错误频率增加了5至12倍。相对于每种聚合酶响应ENU损伤产生的同聚体（重复）序列，异聚体序列处移码突变比例的增加具有统计学意义。对于聚合酶α-引发酶，模板鸟嘌呤残基处的单碱基缺失错误是第二常见的突变事件，其观察频率仅比G→A转换频率低四倍。在聚合酶β反应中，使用烷基化模板时，同聚体（重复）序列处的插入错误频率相对于溶剂对照增加了六倍。这种插入错误的频率仅比聚合酶β的G→A转换错误频率低三倍。尽管ENU通常被认为是一种强效的碱基置换诱变剂，但这些数据表明单功能烷基化剂能够在体外诱导移码突变。烷基化诱导的移码突变发生在重复和非重复DNA序列中；然而，突变特异性取决于DNA聚合酶。
