Shimmoto Michie, Matsumoto Seiji, Odagiri Yukari, Noguchi Eishi, Russell Paul, Masai Hisao
Genome Dynamics Project, Tokyo Metropolitan Institute of Medical Science, Tokyo 113-8613, Japan.
Genes Cells. 2009 Jun;14(6):669-82. doi: 10.1111/j.1365-2443.2009.01300.x. Epub 2009 Apr 30.
The Swi1-Swi3 replication fork protection complex and Mrc1 protein are required for stabilization of stalled replication forks in fission yeast. Hsk1 kinase also plays roles in checkpoint responses elicited by arrested replication forks. We show that both Swi1 and Swi3, the abundance of which are interdependent, are required for chromatin association of Mrc1. Co-immunoprecipitation experiments show the interactions of Swi1-Swi3, Mrc1 and Hsk1. Mrc1 interacts with Swi3 and Hsk1 proteins through its central segment (378-879) containing a SQ/TQ cluster, and this segment is sufficient for checkpoint reaction. The SQ/TQ cluster segment (536-673) is essential but not sufficient for the interactions and for resistance to replication inhibitor hydroxyurea. Mrc1 protein level is increased in hsk1-89 cells due to apparent stabilization, and we have identified a potential phosphodegron sequence. These results suggest that interactions of the Swi1-Swi3 complex and Hsk1 kinase with Mrc1 may play a role in cellular responses to stalled replication forks in fission yeast.
Swi1-Swi3复制叉保护复合体和Mrc1蛋白是裂殖酵母中稳定停滞复制叉所必需的。Hsk1激酶在停滞复制叉引发的检查点反应中也发挥作用。我们发现,Swi1和Swi3的丰度相互依赖,它们都是Mrc1与染色质结合所必需的。免疫共沉淀实验表明了Swi1-Swi3、Mrc1和Hsk1之间的相互作用。Mrc1通过其包含SQ/TQ簇的中央区段(378-879)与Swi3和Hsk1蛋白相互作用,并且该区段足以引发检查点反应。SQ/TQ簇区段(536-673)对于相互作用和对复制抑制剂羟基脲的抗性是必需的,但并不充分。由于明显的稳定性,hsk1-89细胞中的Mrc1蛋白水平升高,并且我们已经鉴定出一个潜在的磷酸化降解序列。这些结果表明,Swi1-Swi3复合体和Hsk1激酶与Mrc1的相互作用可能在裂殖酵母对停滞复制叉的细胞反应中发挥作用。