Patel S, Jazrawi E, Creighton A M, Austin C A, Fisher L M
Molecular Genetics Group, Department of Biochemistry and Immunology, St. George's Hospital Medical School, University of London, London, United Kingdom.
Mol Pharmacol. 2000 Sep;58(3):560-8. doi: 10.1124/mol.58.3.560.
Topoisomerase II is an ATP-operated protein clamp that captures a DNA helix and transports it through another DNA duplex, allowing chromosome segregation at mitosis. A number of cytotoxic bisdioxopiperazines such as ICRF-193 target topoisomerase II by binding and trapping the closed enzyme clamp. To investigate this unusual mode of action, we have used yeast to select plasmid-borne human topoisomerase IIalpha alleles resistant to ICRF-193. Mutations in topoisomerase IIalpha of Leu-169 to Phe (L169F) (in the N-terminal ATPase domain) and Ala-648 to Pro (A648P) (in the core domain) were identified as conferring >50-fold and 5-fold resistance to ICRF-193 in vivo, respectively. The L169F mutation, located next to the Walker A box ATP-binding sequence, resulted in a mutant enzyme displaying ICRF-193-resistant topoisomerase and ATPase activities and whose closed clamp was refractory to ICRF-193-mediated trapping as an annulus on closed circular DNA. These data imply that the mutation interferes directly with ICRF-193 binding to the N-terminal ATPase gate. In contrast, the A648P enzyme displayed topoisomerase activities exhibiting wild-type sensitivity to ICRF-193. We suggest that the inefficient trapping of the A648P closed clamp results either from the observed increased ATP requirement, or more likely, from lowered salt stability, perhaps involving destabilization of ICRF-193 interactions with the B'-B' interface in the core domain. These results provide evidence for at least two different phenotypic classes of ICRF-193 resistance mutations and suggest that bisdioxopiperazine action involves the interplay of both the ATPase and core domains of topoisomerase IIalpha.
拓扑异构酶II是一种由ATP驱动的蛋白质夹子，它捕获一条DNA螺旋并使其穿过另一条DNA双链，从而在有丝分裂时实现染色体分离。许多细胞毒性双二氧哌嗪类药物，如ICRF-193，通过结合并捕获闭合的酶夹子来靶向拓扑异构酶II。为了研究这种不同寻常的作用方式，我们利用酵母筛选出对ICRF-193具有抗性的质粒携带的人拓扑异构酶IIα等位基因。拓扑异构酶IIα中Leu-169突变为Phe（L169F）（位于N端ATP酶结构域）以及Ala-648突变为Pro（A648P）（位于核心结构域）的突变被鉴定为分别在体内对ICRF-193具有大于50倍和5倍的抗性。位于沃克A框ATP结合序列旁边的L169F突变导致一种突变酶表现出对ICRF-193具有抗性的拓扑异构酶和ATP酶活性，并且其闭合夹子作为闭环DNA上的环对ICRF-193介导的捕获具有抗性。这些数据表明该突变直接干扰了ICRF-193与N端ATP酶门的结合。相比之下，A648P酶表现出对ICRF-193具有野生型敏感性的拓扑异构酶活性。我们认为，A648P闭合夹子捕获效率低下要么是由于观察到的ATP需求增加，要么更可能是由于盐稳定性降低，可能涉及ICRF-193与核心结构域中B'-B'界面相互作用的不稳定。这些结果为ICRF-193抗性突变的至少两种不同表型类别提供了证据，并表明双二氧哌嗪的作用涉及拓扑异构酶IIα的ATP酶和核心结构域的相互作用。
