Feig D I, Loeb L A
Department of Biochemistry, Joseph Gottstein Memorial Cancer Research Laboratory, University of Washington, Seattle 98195.
Biochemistry. 1993 Apr 27;32(16):4466-73. doi: 10.1021/bi00067a040.
Reactive oxygen species, produced in cells by a variety of mechanisms, damage DNA and cause mutations. To characterize the types of mutations produced in mammalian cells, we copied DNA damaged by reactive oxygen species with mammalian DNA polymerase beta. Double-stranded circular M13mp2 DNA containing a 361-nucleotide single-stranded gap within the lacZ gene was damaged by aerobic incubation with Fe2+ and H2O2. The gap then was filled by purified recombinant rat DNA polymerase beta, and the DNA was transfected into Escherichia coli. Mutations within the nonessential lacZ gene for beta-galactosidase were identified by reduced alpha-complementation. In this system, oxidative damage increased the mutation frequency within the target region by an average of 4.3-fold. At certain sites, the base substitution rate is nearly 300 times greater than would be expected to result from a random distribution of damage. The oxidatively induced mutations fall into two categories: those apparently caused by direct miscoding of modified DNA and those associated with enhanced misincorporation at prexisting polymerase-specific hot spots. The latter group may be due to a conformational change in the DNA caused by oxidative modification and could be indicative of a novel mutagenic mechanism.
细胞通过多种机制产生的活性氧会损伤DNA并导致突变。为了表征哺乳动物细胞中产生的突变类型，我们用哺乳动物DNA聚合酶β复制了被活性氧损伤的DNA。含有位于lacZ基因内361个核苷酸单链缺口的双链环状M13mp2 DNA通过与Fe2+和H2O2进行需氧孵育而受损。然后用纯化的重组大鼠DNA聚合酶β填补缺口，并将DNA转染到大肠杆菌中。通过降低α互补来鉴定β-半乳糖苷酶非必需lacZ基因内的突变。在该系统中，氧化损伤使靶区域内的突变频率平均增加了4.3倍。在某些位点，碱基替换率比随机分布损伤所预期的高出近300倍。氧化诱导的突变分为两类：一类显然是由修饰DNA的直接错配编码引起的，另一类与在先前存在的聚合酶特异性热点处增强的错配掺入有关。后一组可能是由于氧化修饰导致DNA构象变化引起的，可能表明存在一种新的诱变机制。
