Feig D I, Loeb L A
Joseph Gottstein Memorial Cancer Research Laboratory, Department of Biochemistry, University of Washington, Seattle 98195.
J Mol Biol. 1994 Jan 7;235(1):33-41. doi: 10.1016/s0022-2836(05)80009-9.
Oxygen free radicals are produced in large amounts by normal cellular processes. Damage to DNA by these reactive species has been implicated in mutagenesis and may be important in the etiology of a variety of human diseases. In this study we investigate the types of mutations produced in vitro as a result of DNA damage by oxygen free radicals. We used a lacZ alpha forward mutation assay in which M13 viral DNA is damaged in vitro, replicated with purified DNA polymerase alpha or beta, transfected into E. coli, and screened for mutations by reduced alpha-complementation of beta-galactosidase activity. By determining the effects of damaged templates on the fidelity of individual DNA polymerases involved in replication and repair, we address the role of specific DNA polymerases in mutagenesis induced by reactive oxygen species. Aerobic incubation of DNA with 100 microM CuCl, 10 microM H2O2 and 100 microM ascorbic acid results in a 3.3-fold and a 3.6-fold elevation in mutation frequency for polymerases alpha and beta, respectively. The specificity and location of the induced mutations, however, are entirely different. For polymerase alpha, A to C, and C to A transversions and deletions of C are each elevated more than 10-fold over their frequencies on undamaged template. For polymerase beta, A to T, C to T, C to A, G to C, and G to T substitutions, and deletions of G are elevated by damage. The frequency of mutants containing two or more closely spaced substitutions is also markedly increased by template damage although the types of mutations and their positions are again specific to each DNA polymerase. We conclude that, for oxidative lesions, the frequency and the types of mutations are determined in part by the DNA polymerase that encounters the site of damage.
正常细胞过程会大量产生氧自由基。这些活性物质对DNA的损伤与诱变作用有关，可能在多种人类疾病的病因学中起重要作用。在本研究中，我们调查了氧自由基导致DNA损伤在体外产生的突变类型。我们使用了一种lacZα正向突变试验，其中M13病毒DNA在体外被损伤，用纯化的DNA聚合酶α或β进行复制，转染到大肠杆菌中，并通过β-半乳糖苷酶活性的α-互补降低来筛选突变。通过确定受损模板对参与复制和修复的单个DNA聚合酶保真度的影响，我们探讨了特定DNA聚合酶在活性氧诱导的诱变中的作用。将DNA与100微摩尔氯化铜、10微摩尔过氧化氢和100微摩尔抗坏血酸进行需氧孵育，结果聚合酶α和β的突变频率分别升高了3.3倍和3.6倍。然而，诱导突变的特异性和位置完全不同。对于聚合酶α，A到C、C到A的颠换以及C的缺失在频率上比未受损模板上各自升高了10倍以上。对于聚合酶β，A到T、C到T、C到A、G到C和G到T的替换以及G的缺失因损伤而升高。尽管突变类型及其位置再次因每种DNA聚合酶而异，但含有两个或更多紧密间隔替换的突变体频率也因模板损伤而显著增加。我们得出结论，对于氧化性损伤，突变的频率和类型部分由遇到损伤位点的DNA聚合酶决定。
