Kaplun L, Ivantsiv Y, Kornitzer D, Raveh D
Life Sciences Department, Ben Gurion University of the Negev, Box 653, Beersheba 84105, Israel.
Proc Natl Acad Sci U S A. 2000 Aug 29;97(18):10077-82. doi: 10.1073/pnas.97.18.10077.
Ho endonuclease of Saccharomyces cerevisiae is a homing endonuclease that makes a site-specific double-strand break in the MAT gene in late G(1). Here we show that Ho is rapidly degraded via the ubiquitin-26S proteasome system through two ubiquitin-conjugating enzymes UBC2(Rad6) and UBC3(Cdc34). UBC2(Rad6) is complexed with the ring finger DNA-binding protein Rad18, and we find that Ho is stabilized in rad18 mutants. We show that the Ho degradation pathway involving UBC3(Cdc34) goes through the Skp1/Cdc53/F-box (SCF) ubiquitin ligase complex and identify a F-box protein, Yml088w, that is required for Ho degradation. Components of a defined pathway of the DNA damage response, MEC1, RAD9, and CHK1, are also necessary for Ho degradation, whereas functions of the RAD24 epistasis group and the downstream effector RAD53 have no role in degradation of Ho. Our results indicate a link between the endonuclease function of Ho and its destruction.
酿酒酵母的Ho核酸内切酶是一种归巢核酸内切酶,它在G(1)晚期在MAT基因中产生位点特异性双链断裂。在这里,我们表明Ho通过泛素-26S蛋白酶体系统,经由两种泛素结合酶UBC2(Rad6)和UBC3(Cdc34)迅速降解。UBC2(Rad6)与环指DNA结合蛋白Rad18复合,我们发现Ho在rad18突变体中稳定存在。我们表明,涉及UBC3(Cdc34)的Ho降解途径通过Skp1/Cdc53/F-box(SCF)泛素连接酶复合物,并鉴定出一种F-box蛋白Yml088w,它是Ho降解所必需的。DNA损伤反应特定途径的组成部分MEC1、RAD9和CHK1对于Ho降解也是必需的,而RAD24上位性组和下游效应物RAD53的功能在Ho降解中不起作用。我们的结果表明Ho的核酸内切酶功能与其破坏之间存在联系。