Abstract：
The CMG helicase is the stable core of the eukaryotic replisome and is ubiquitylated and disassembled during DNA replication termination. Fungi and animals use different enzymes to ubiquitylate the Mcm7 subunit of CMG, suggesting that CMG ubiquitylation arose repeatedly during eukaryotic evolution. Until now, it was unclear whether cells also have ubiquitin-independent pathways for helicase disassembly and whether CMG disassembly is essential for cell viability. Using reconstituted assays with budding yeast CMG, we generated the mcm7-10R allele that compromises ubiquitylation by SCFDia2. mcm7-10R delays helicase disassembly in vivo, driving genome instability in the next cell cycle. These data indicate that defective CMG ubiquitylation explains the major phenotypes of cells lacking Dia2. Notably, the viability of mcm7-10R and dia2∆ is dependent upon the related Rrm3 and Pif1 DNA helicases that have orthologues in all eukaryotes. We show that Rrm3 acts during S-phase to disassemble old CMG complexes from the previous cell cycle. These findings indicate that CMG disassembly is essential in yeast cells and suggest that Pif1-family helicases might have mediated CMG disassembly in ancestral eukaryotes.
摘要：
CMG解旋酶是真核复制体的稳定核心，在DNA复制终止过程中被泛素化和分解。真菌和动物使用不同的酶来泛素化CMG的Mcm7亚基，表明CMG泛素化在真核进化过程中反复出现。直到现在,目前尚不清楚细胞是否也有不依赖泛素的解旋酶分解途径,以及CMG分解是否对细胞活力至关重要.使用含芽酵母CMG的重组测定法，我们产生了mcm7-10R等位基因，该等位基因损害了SCFDia2的泛素化。mcm7-10R在体内延迟解旋酶分解，在下一个细胞周期中驱动基因组不稳定。这些数据表明有缺陷的CMG泛素化解释了缺乏Dia2的细胞的主要表型。值得注意的是,mcm7-10R和dia2的活力取决于在所有真核生物中具有直系同源物的相关Rrm3和Pif1DNA解旋酶。我们表明，Rrm3在S期起作用，可以从先前的细胞周期中分解旧的CMG复合物。这些发现表明CMG拆解在酵母细胞中是必不可少的，并表明Pif1家族解旋酶可能在祖先真核生物中介导了CMG拆解。
