D'Arpa P, Liu L F
Department of Pharmacology, University of Medicine and Dentistry of New Jersey-Robert Wood Johnson Medical School, Piscataway 08854.
Exp Cell Res. 1995 Mar;217(1):125-31. doi: 10.1006/excr.1995.1071.
Eukaryotic DNA topoisomerase I has been recently shown to be associated with the transcriptional machinery and has also been implicated to function in DNA replication and perhaps other DNA transactions. We have identified several differentially phosphorylated forms of mammalian topoisomerase I as electrophoretic variants by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. These differently phosphorylated forms cleave chromosomal DNA in cells and also relax supercoiled DNA with about equal activity, suggesting that they primarily function other than to activate catalysis. One of the phosphorylated forms is specifically present during mitosis. Upon transition from mitosis into G1 phase, two forms differing in phosphorylation state appear and persist throughout the remainder of interphase. When cells are incubated in a pellet, one of the interphase phosphorylated forms disappears coincidentally with an increase in the abundance of the other; if the cell pellet is disrupted and the cells are reincubated in suspension, the forms rapidly shift back to their original abundance levels. Finally, a shift in relative abundance of the differently phosphorylated interphase forms is observed when transcription is inhibited. These results suggest that dynamic phosphorylation and dephosphorylation regulate topoisomerase I during RNA transcription and cell cycle progression.
真核生物DNA拓扑异构酶I最近被证明与转录机制相关，并且也被认为在DNA复制以及可能的其他DNA事务中发挥作用。我们通过十二烷基硫酸钠-聚丙烯酰胺凝胶电泳将哺乳动物拓扑异构酶I的几种差异磷酸化形式鉴定为电泳变体。这些不同磷酸化形式在细胞中切割染色体DNA，并且以大致相同的活性松弛超螺旋DNA，这表明它们的主要功能并非激活催化作用。其中一种磷酸化形式在有丝分裂期间特异性存在。从有丝分裂过渡到G1期时，出现两种磷酸化状态不同的形式，并在间期的其余时间持续存在。当细胞在沉淀中孵育时，其中一种间期磷酸化形式消失，同时另一种的丰度增加；如果细胞沉淀被破坏并且细胞在悬浮液中重新孵育，这些形式会迅速恢复到它们原来的丰度水平。最后，当转录受到抑制时，观察到不同磷酸化的间期形式的相对丰度发生变化。这些结果表明，动态磷酸化和去磷酸化在RNA转录和细胞周期进程中调节拓扑异构酶I。
