Maric Marija, Mukherjee Progya, Tatham Michael H, Hay Ronald, Labib Karim
MRC Protein Phosphorylation and Ubiquitylation Unit, Sir James Black Centre, School of Life Sciences, University of Dundee, Dow Street, Dundee DD1 5EH, UK.
Gene Regulation and Expression Division, School of Life Sciences, University of Dundee, Dow Street, Dundee DD1 5EH, UK.
Cell Rep. 2017 Mar 28;18(13):3033-3042. doi: 10.1016/j.celrep.2017.03.020.
Disassembly of the Cdc45-MCM-GINS (CMG) DNA helicase is the key regulated step during DNA replication termination in eukaryotes, involving ubiquitylation of the Mcm7 helicase subunit, leading to a disassembly process that requires the Cdc48 "segregase". Here, we employ a screen to identify partners of budding yeast Cdc48 that are important for disassembly of ubiquitylated CMG helicase at the end of chromosome replication. We demonstrate that the ubiquitin-binding Ufd1-Npl4 complex recruits Cdc48 to ubiquitylated CMG. Ubiquitylation of CMG in yeast cell extracts is dependent upon lysine 29 of Mcm7, which is the only detectable site of ubiquitylation both in vitro and in vivo (though in vivo other sites can be modified when K29 is mutated). Mutation of K29 abrogates in vitro recruitment of Ufd1-Npl4-Cdc48 to the CMG helicase, supporting a model whereby Ufd1-Npl4 recruits Cdc48 to ubiquitylated CMG at the end of chromosome replication, thereby driving the disassembly reaction.
Cdc45-MCM-GINS（CMG）DNA解旋酶的拆卸是真核生物DNA复制终止过程中的关键调控步骤，涉及Mcm7解旋酶亚基的泛素化，导致一个需要Cdc48“分离酶”的拆卸过程。在这里，我们通过筛选来鉴定出芽酵母Cdc48的伙伴，这些伙伴对于在染色体复制末端拆卸泛素化的CMG解旋酶很重要。我们证明，泛素结合蛋白Ufd1-Npl4复合物将Cdc48招募到泛素化的CMG上。酵母细胞提取物中CMG的泛素化依赖于Mcm7的赖氨酸29，这是体外和体内唯一可检测到的泛素化位点（尽管在体内当K29发生突变时其他位点也可被修饰）。K29的突变消除了体外Ufd1-Npl4-Cdc48对CMG解旋酶的招募，支持了一种模型，即Ufd1-Npl4在染色体复制末端将Cdc48招募到泛素化的CMG上，从而驱动拆卸反应。
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