Povirk L F, Bennett R A, Wang P, Swerdlow P S, Austin M J
Department of Pharmacology and Toxicology, Medical College of Virginia, Virginia Commonwealth University, Richmond 23298.
J Mol Biol. 1994 Oct 21;243(2):216-26. doi: 10.1006/jmbi.1994.1649.
One possible mechanism for the generation of deletion mutations is inaccurate repair of DNA double-strand breaks. In an attempt to detect such aberrant repair events in intact cells, confluent stationary phase cultures of chinese hamster ovary D422 cells, which are hemizygous for aprt, were treated for two days with low concentrations of bleomycin, and aprt mutant clones were selected and analyzed by polymerase chain reaction and DNA sequencing. Bleomycin was quite mutagenic in stationary phase cells, increasing the mutant frequency by five to 40-fold at 5 to 50% survival. While spontaneous mutations generated under these conditions were predominantly base substitutions, the majority of the bleomycin-induced mutations were very small deletions, with lesser numbers of large deletions/rearrangements and base substitutions. Although the small deletions tended to be clustered in several short segments of the gene, nucleosome positioning studies indicated that there was no consistent phasing of nucleosomes in aprt, suggesting that the clustering was due to sequence specificity rather than chromatin structure. About half of the bleomycin-induced mutations were single-base-pair (-1) deletions, and the majority of these involved deletion of one C in a G-Cn sequence (n > or = 2). At such sites, bleomycin is known to induce double-strand breaks by fragmentation of deoxyribose moieties at the same sequence position in both strands, resulting in a blunt-ended double-strand break with 5'-phosphate and 3'-phosphoglycolate termini. Thus, this sequence specificity is consistent with a model in which bleomycin-induced -1 deletions are generated by a double-strand break rejoining process involving removal of phosphoglycolate moieties from both 3' ends, followed by blunt-end ligation. The results support the view that repair of free radical-mediated double-strand breaks in mammalian cells in G1/G0 phase can be effected by such simple end-joining mechanisms, without the need for homologous recombination.
缺失突变产生的一种可能机制是DNA双链断裂的不准确修复。为了在完整细胞中检测此类异常修复事件，对中国仓鼠卵巢D422细胞（该细胞为aprt半合子）的汇合静止期培养物用低浓度博来霉素处理两天，然后选择aprt突变克隆并通过聚合酶链反应和DNA测序进行分析。博来霉素在静止期细胞中具有很强的诱变作用，在5%至50%存活率时，使突变频率增加了5至40倍。虽然在这些条件下产生的自发突变主要是碱基替换，但博来霉素诱导的突变大多数是非常小的缺失，大缺失/重排和碱基替换的数量较少。尽管小缺失倾向于聚集在基因的几个短片段中，但核小体定位研究表明，aprt中核小体没有一致的相位，这表明聚集是由于序列特异性而非染色质结构。大约一半的博来霉素诱导突变是单碱基对（-1）缺失，其中大多数涉及G-Cn序列（n≥2）中一个C的缺失。在这些位点，已知博来霉素通过切割两条链上相同序列位置的脱氧核糖部分来诱导双链断裂，从而产生具有5'-磷酸和3'-磷酸乙醇酸末端的平端双链断裂。因此，这种序列特异性与一个模型一致，在该模型中，博来霉素诱导的-1缺失是由双链断裂重新连接过程产生的，该过程涉及从两个3'末端去除磷酸乙醇酸部分，然后进行平端连接。这些结果支持这样一种观点，即G1/G0期哺乳动物细胞中自由基介导的双链断裂的修复可以通过这种简单的末端连接机制实现，而无需同源重组。
