Wang P, Zhou R H, Zou Y, Jackson-Cook C K, Povirk L F
Department of Pharmacology and Toxicology, Medical College of Virginia, Virginia Commonwealth University, Richmond, VA 23298, USA.
Proc Natl Acad Sci U S A. 1997 Oct 28;94(22):12018-23. doi: 10.1073/pnas.94.22.12018.
Chromosomal translocations induced by ionizing radiation and radiomimetic drugs are thought to arise by incorrect joining of DNA double-strand breaks. To dissect such misrepair events at a molecular level, large-scale, bleomycin-induced rearrangements in the aprt gene of Chinese hamster ovary D422 cells were mapped, the breakpoints were sequenced, and the original non-aprt parental sequences involved in each rearrangement were recovered from nonmutant cells. Of seven rearrangements characterized, six were reciprocal exchanges between aprt and unrelated sequences. Consistent with a mechanism involving joining of exchanged double-strand break ends, there was, in most cases, no homology between the two parental sequences, no overlap in sequences retained at the two newly formed junctions, and little or no loss of parental sequences (usually </=2 bp) at the breakpoints. The breakpoints were strongly correlated (P < 0.0001) with expected sites of bleomycin-induced, double-strand breaks. Fluorescence in situ hybridization indicated that, in six of the mutants, the rearrangement was accompanied by a chromosomal translocation at the aprt locus, because upstream and downstream flanking sequences were detected on separate chromosomes. The results suggest that repair of free radical-mediated, double-strand breaks in confluence-arrested cells is effected by a conservative, homology-independent, end-joining pathway that does not involve single-strand intermediate and that misjoining of exchanged ends by this pathway can directly result in chromosomal translocations.
电离辐射和拟放射性药物诱导的染色体易位被认为是由DNA双链断裂的错误连接引起的。为了在分子水平上剖析此类错误修复事件，对中国仓鼠卵巢D422细胞的aprt基因中博来霉素诱导的大规模重排进行了定位，对断点进行了测序，并从非突变细胞中回收了每个重排中涉及的原始非aprt亲本序列。在已表征的七个重排中，六个是aprt与不相关序列之间的相互交换。与涉及交换的双链断裂末端连接的机制一致，在大多数情况下，两个亲本序列之间没有同源性，在两个新形成的连接处保留的序列没有重叠，并且在断点处亲本序列几乎没有丢失（通常≤2 bp）。断点与博来霉素诱导的双链断裂的预期位点高度相关（P < 0.0001）。荧光原位杂交表明，在六个突变体中，重排伴随着aprt位点的染色体易位，因为在上游和下游侧翼序列在不同的染色体上被检测到。结果表明，汇合停滞细胞中自由基介导的双链断裂的修复是通过一种保守的、不依赖同源性的末端连接途径实现的，该途径不涉及单链中间体，并且该途径中交换末端的错误连接可直接导致染色体易位。
