Nucleotide-resolution mapping of topoisomerase-mediated and apoptotic DNA strand scissions at or near an MLL translocation hotspot
Nucleotide-resolution mapping of topoisomerase-mediated and apoptotic DNA strand scissions at or near an MLL translocation hotspot
复制标题
DOI：
10.1086/507791
复制
发表时间：
2006-11-01
影响因子：
9.8
通讯作者：
Tremblay, Alain
中科院分区：
文献类型：
--
作者：
Mirault, Marc-Edouard;Boucher, Patrick;Tremblay, Alain
治疗相关性急性髓细胞白血病（t-AML）的出现与DNA拓扑异构酶II（TOP 2）靶向药物治疗和染色体易位（通常涉及MLL或ALL-1基因）有关。两种不同的机制被认为是t-AML易位的潜在触发因素：TOP2介导的DNA切割和凋亡的高阶染色质片段化。TOP2在这一过程中的作用的评估一直受到阻碍，缺乏技术，允许在体内映射的TOP2介导的DNA切割在单拷贝基因的核苷酸分辨率。一种新的方法，延伸连接介导的聚合酶链反应（ELMPCR），在这里被用来映射拓扑异构酶介导的DNA链断裂和凋亡DNA切割跨MLL在人类细胞中的易易位区域。我们报告的第一个基因组图谱整合易位断点和拓扑异构酶I，TOP 2，和凋亡DNA裂解位点在核苷酸分辨率跨越MLL区域窝藏t-AML易位热点。该热点的侧翼是一个TOP 2切割位点，并定位在一个小的凋亡切割区，其中多个单，双链断裂诱导的半胱天冬酶激活的凋亡核酸酶的一端。这种切割模式与在外显子12区域下游200 bp处观察到的切割模式形成鲜明对比，后者显示出更强的凋亡切割，但未检测到双链断裂，也未报告t-AML相关断裂点。t-AML断裂点的定位和显著的聚集不能简单地用DNA切割模式来解释，但可能是由于TOP2中毒、凋亡DNA切割和逃避凋亡细胞中高阶染色质结构特定位点的DNA修复尝试之间的潜在相互作用。ELMPCR为研究DNA拓扑异构酶在与涉及拓扑异构酶靶向药物的癌症治疗相关的基本遗传过程和易位中的作用提供了新的工具。
The emergence of therapy-related acute myeloid leukemia (t-AML) has been associated with DNA topoisomerase II (TOP2)-targeted drug treatments and chromosomal translocations frequently involving the MLL, or ALL-1, gene. Two distinct mechanisms have been implicated as potential triggers of t-AML translocations: TOP2-mediated DNA cleavage and apoptotic higher-order chromatin fragmentation. Assessment of the role of TOP2 in this process has been hampered by a lack of techniques allowing in vivo mapping of TOP2-mediated DNA cleavage at nucleotide resolution in single-copy genes. A novel method, extension ligation-mediated polymerase chain reaction (ELMPCR), was used here for mapping topoisomerase-mediated DNA strand breaks and apoptotic DNA cleavage across a translocation-prone region of MLL in human cells. We report the first genomic map integrating translocation breakpoints and topoisomerase I, TOP2, and apoptotic DNA cleavage sites at nucleotide resolution across an MLL region harboring a t-AML translocation hotspot. This hotspot is flanked by a TOP2 cleavage site and is localized at one extremity of a minor apoptotic cleavage region, where multiple single- and double-strand breaks were induced by caspase-activated apoptotic nucleases. This cleavage pattern was in sharp contrast to that observed similar to 200 bp downstream in the exon 12 region, which displayed much stronger apoptotic cleavage but where no double-strand breaks were detected and no t-AML-associated breakpoints were reported. The localization and remarkable clustering of the t-AML breakpoints cannot be explained simply by the DNA cleavage patterns but might result from potential interactions between TOP2 poisoning, apoptotic DNA cleavage, and DNA repair attempts at specific sites of higher-order chromatin structure in apoptosis-evading cells. ELMPCR provides a new tool for investigating the role of DNA topoisomerases in fundamental genetic processes and translocations associated with cancer treatments involving topoisomerase-targeted drugs.