Near-precise interchromosomal recombination and functional DNA topoisomerase II cleavage sites at MLL and AF-4 genomic breakpoints in treatment-related acute lymphoblastic leukemia with t(4;11) translocation
Near-precise interchromosomal recombination and functional DNA topoisomerase II cleavage sites at MLL and AF-4 genomic breakpoints in treatment-related acute lymphoblastic leukemia with t(4;11) translocation
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DOI：
10.1073/pnas.171309898
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发表时间：
2001-08-14
影响因子：
11.1
通讯作者：
Felix, CA
中科院分区：
文献类型：
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作者：
Lovett, BD;Lo Nigro, L;Felix, CA
我们通过分子和生物化学方法分析了一位曾服用依托泊苷和更生霉素的白血病患者t（4;11）的der（11）和der（4）基因组断点连接，以了解易位机制和相关药物暴露。通过PCR扩增基因组断点连接。在体外与人DNA拓扑异构酶II α和依托泊苷、依托泊苷儿茶酚、依托泊苷醌或更生霉素孵育后，检查含有MLL和AF-4易位断裂点的正常同系物的DNA底物的切割。der（11）和der（4）基因组断裂点连接均涉及MLL内含子6和AF-4内含子3。除了单个模板化核苷酸的总体增益之外，在序列水平上的扩增是精确的。MLL和AF-4的易位断裂点为DNA拓扑异构酶11的切割位点。依托泊苷及其代谢产物，但不是放线菌素dactinomycin，增强裂解在这些网站。假设DNA拓扑异构酶11是断裂的介质，在连接末端以产生观察到的基因组断裂点连接之前，需要处理由DNA拓扑异构酶11诱导的交错切口，包括核酸外切酶缺失和模板指导的聚合。这些数据与涉及通过DNA拓扑异构酶11亚基的简单交换和DNA链转移的染色体间重组的易位机制不一致;然而，与MLL和AF-4中的相互DNA拓扑异构酶11切割事件一致，其中两个断裂变得稳定，DNA末端被加工并经历连接。依托泊苷和/或其代谢产物（而非更生霉素）可能是该患者的相关暴露。
We analyzed the der(11) and der(4) genomic breakpoint junctions of a t(4;11) in the leukemia of a patient previously administered etoposide and dactinomycin by molecular and biochemical approaches to gain insights about the translocation mechanism and the relevant drug exposure. The genomic breakpoint junctions were amplified by PCR. Cleavage of DNA substrates containing the normal homologues of the MLL and AF-4 translocation breakpoints was examined in vitro upon incubation with human DNA topoisomerase II alpha and etoposide, etoposide catechol, etoposide quinone, or dactinomycin. The der(11) and der(4) genomic breakpoint junctions both involved MLL intron 6 and AF-4 intron 3. Recombination was precise at the sequence level except for the overall gain of a single templated nucleotide. The translocation breakpoints in MLL and AF-4 were DNA topoisomerase 11 cleavage sites. Etoposide and its metabolites, but not dactinomycin, enhanced cleavage at these sites. Assuming that DNA topoisomerase 11 was the mediator of the breakage, processing of the staggered nicks induced by DNA topoisomerase 11, including exonucleolytic deletion and template-directed polymerization, would have been required before ligation of the ends to generate the observed genomic breakpoint junctions. These data are inconsistent with a translocation mechanism involving interchromosomal recombination by simple exchange of DNA topoisomerase 11 subunits and DNA-strand transfer; however, consistent with reciprocal DNA topoisomerase 11 cleavage events in MLL and AF-4 in which both breaks became stable, the DNA ends were processed and underwent ligation. Etoposide and/or its metabolites, but not dactinomycin, likely were the relevant exposures in this patient.