Phosphorylation of CMG helicase and Tof1 is required for programmed fork arrest
Phosphorylation of CMG helicase and Tof1 is required for programmed fork arrest
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DOI：
10.1073/pnas.1607552113
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发表时间：
2016-06-28
影响因子：
11.1
通讯作者：
O'Donnell, Michael E.
中科院分区：
文献类型：
--
作者：
Bastia, Deepak;Srivastava, Pankaj;O'Donnell, Michael E.
几个重要的生理事务，包括复制寿命（RLS）的控制，复制和转录之间的冲突，和细胞分化的预防，需要程序化的复制叉停滞（PFA）。然而，PFA的一般机制仍然难以捉摸。我们以前表明，Tof 1-Csm 3叉保护复合物是必不可少的PFA通过拮抗Rrm 3解旋酶，取代非组蛋白蛋白障碍，阻碍叉进展。在这里，我们表明，Dbf 4依赖性激酶（DDK）的酿酒酵母，而不是其他DNA复制因子的突变，大大减少了PFA在复制叉的障碍在核糖体DNA阵列的间隔区。DDK的一个关键靶标是微型染色体维持（Mcm）2-7复合物，已知其需要DDK磷酸化以形成活性CMG [Cdc 45（细胞分裂周期基因45）、Mcm 2 -7、GINS（Go、Ichi、Ni和San）]解旋酶。体内实验表明，DDK的突变失活导致Tof 1从染色质组分中释放。体外结合实验证实，CMG和/或Mcm 2 -7必须被磷酸化以结合磷酸-Tof 1-Csm 3，而不是其去磷酸化形式。抑制突变绕过Mcm 2 -7磷酸化的DDK恢复PFA的激酶的情况下的要求。Tof 1在染色质组分和PFA在体内的保留促进了抑制剂mcm 5-bob 1，绕过DDK的要求，表明在这种条件下的激酶，而不是DDK催化Tof 1的磷酸化。我们建议，磷酸化调节的招聘和保留Tof 1-Csm 3的复制体，这种复杂的拮抗Rrm 3解旋酶，从而促进PFA，通过保持完整的Fob 1-Ter复合物。
Several important physiological transactions, including control of replicative life span (RLS), prevention of collision between replication and transcription, and cellular differentiation, require programmed replication fork arrest (PFA). However, a general mechanism of PFA has remained elusive. We previously showed that the Tof1-Csm3 fork protection complex is essential for PFA by antagonizing the Rrm3 helicase that displaces nonhistone protein barriers that impede fork progression. Here we show that mutations of Dbf4-dependent kinase (DDK) of Saccharomyces cerevisiae, but not other DNA replication factors, greatly reduced PFA at replication fork barriers in the spacer regions of the ribosomal DNA array. A key target of DDK is the mini chromosome maintenance (Mcm) 2-7 complex, which is known to require phosphorylation by DDK to form an active CMG [Cdc45 (cell division cycle gene 45), Mcm2-7, GINS (Go, Ichi, Ni, and San)] helicase. In vivo experiments showed that mutational inactivation of DDK caused release of Tof1 from the chromatin fractions. In vitro binding experiments confirmed that CMG and/or Mcm2-7 had to be phosphorylated for binding to phospho-Tof1-Csm3 but not to its dephosphorylated form. Suppressor mutations that bypass the requirement for Mcm2-7 phosphorylation by DDK restored PFA in the absence of the kinase. Retention of Tof1 in the chromatin fraction and PFA in vivo was promoted by the suppressor mcm5-bob1, which bypassed DDK requirement, indicating that under this condition a kinase other than DDK catalyzed the phosphorylation of Tof1. We propose that phosphorylation regulates the recruitment and retention of Tof1-Csm3 by the replisome and that this complex antagonizes the Rrm3 helicase, thereby promoting PFA, by preserving the integrity of the Fob1-Ter complex.