Genome Instability induced in cancer cells carrying mutations in Type II topoisomerases

DNA拓扑异构酶II（Top2）通过在DNA中造成双链断裂并将DNA链转移到DNA链上来改变DNA拓扑结构。 完整的双链体瞬时双链断裂是通过蛋白质/DNA 称为裂解复合物的共价中间体。哺乳动物细胞含有两种Top2同种型，称为Top2 Top2α和Top2β。这两种酶具有不同的生物学功能，Top2α在其中起关键作用， 染色体分离和Top2β在转录中具有独特的作用。虽然DNA切割机制 允许细胞催化DNA构象的变化，而没有坦率的DNA双链的危险 断裂时，多种机制可干扰酶的切割/再连接平衡。为 例如，抗癌药物如依托泊苷产生升高水平的切割复合物， 细胞毒性和基因组不稳定性。靶向Top2的药物也会导致继发性恶性肿瘤， 酶诱导的DNA损伤。由于Top2可以导致基因组不稳定性，这是癌症的标志， Top2的失败可能是导致癌症的基因组不稳定性的驱动因素。我们发现了独特的突变体， 酵母Top2和两种在没有抑制剂的情况下产生高水平DNA损伤的人类同种型 的酶。这些突变酶有一个独特的特点：虽然它们可以在酵母中表达， 细胞修复DNA损伤，它们在DNA修复缺陷细胞，如rad 52-细胞中的表达 导致细胞死亡。我们检测了这些突变体之一（top2-F1025 Y，R1128 G）是否诱导了 酵母，并发现该等位基因的表达导致一种新的突变特征，其特征在于从头 依赖于DSB修复的非同源末端连接途径的DNA序列的重复。 最近，研究表明，表达Top2α-K743 N的人类癌细胞与肿瘤细胞凋亡相关。 与酵母top2-F1025 Y、R1128 G诱导的突变特征密切相关，表明Top2α- K743 N是一种引起基因组不稳定性的突变子。在这个应用程序中，我们将研究生物化学 Top2α-K743 N的特征。此外，在癌症中也发现了少量的复发性突变 Top2α或Top2β细胞。值得注意的是，Top2β-R656 H已从超过16种不同的肿瘤中回收。 起源我们将应用我们开发的用于研究top2-F1025 Y，R1128 G的工具来研究复发突变， 在Top2α和Top2β中均发现。我们将使用突变酶在rad 52-细胞中的表达和诱导 依托泊苷超敏性作为初步筛选，以确定突变蛋白是否诱导细胞毒性 DNA损伤。将对推定的DNA损伤Top2蛋白进行纯化，并表征其破坏Top2蛋白的能力。 在体外产生DNA损伤。最后，我们将开始产生表达突变体的哺乳动物细胞系 通过CRISPR/Cas9编辑或异位表达来表征突变酶如何导致基因突变， 基因组的不稳定性。我们的研究结果将确定在肿瘤中发现的Top2改变是否可以致癌。

DNA topoisomerase II (Top2) alters DNA topology by making a double-strand break in DNA and passing an intact duplex through the break. The transient double strand break is generated through a protein/DNA covalent intermediate termed the cleavage complex. Mammalian cells contain two Top2 isoforms termed Top2α and Top2β. The two enzymes have distinct biological functions with Top2α playing a key role in chromosome segregation and Top2β having unique roles in transcription. While the DNA cleavage mechanism of Top2 allows cells to catalyze changes in DNA conformation without the dangers of frank DNA double strand breaks, a variety of mechanisms can interfere with the cleavage/religation equilibrium of the enzyme. For example, anti-cancer drugs like etoposide generate elevated levels of cleavage complexes leading to cytotoxicity and genome instability. Drugs targeting Top2 also cause secondary malignancies that arise from enzyme induced DNA damage. Since Top2 can cause genome instability, a hallmark of cancer, it is plausible that Top2 failure could be a driver of genome instability leading to cancer. We identified unique mutants in yeast Top2 and the two human isoforms that generated high levels of DNA damage in the absence of inhibitors of the enzyme. These mutant enzymes have a unique characteristic: while they can be expressed in yeast cells proficient for repairing DNA damage, their expression in DNA repair deficient cells, such as rad52- cells leads to cell killing. We examined whether one of these mutants, (top2-F1025Y,R1128G) induced mutations in yeast and found that expression of this allele led to a novel mutation signature that is characterized by de novo duplications of DNA sequence that depend on the nonhomologous end-joining pathway of DSB repair. Recently, it has been shown that human cancer cells that express Top2α-K743N are associated with a mutational signature closely related to that induced by yeast top2-F1025Y,R1128G suggesting that Top2α- K743N is a mutator that gives rise to genome instability. In this application, we will study the biochemical characteristics of Top2α-K743N. In addition, a small number of recurrent mutations have been found in cancer cells in Top2α or Top2β. Notably, Top2β-R656H has been recovered from more than 16 tumors of diverse origin. We will apply the tools we developed for studying top2-F1025Y,R1128G to the recurrent mutations found in both Top2α and Top2β. We will use expression of the mutant enzymes in rad52- cells and induction of etoposide hypersensitivity as a preliminary screen to determine whether the mutant proteins induce cytotoxic DNA damage. Putative DNA damaging Top2 proteins will be will be purified and characterized for the ability to generate DNA damage in vitro. Finally, we will begin to generate mammalian cell lines that express the mutant alleles by either CRISPR/Cas9 editing or ectopic expression to characterize how the mutant enzymes can lead to genome instability. Our results will establish whether Top2 alterations found in tumors can be oncogenic.