Csm3, Tof1, and Mrc1 Form a Heterotrimeric Mediator Complex That Associates with DNA Replication Forks

Takehiko Itoh,Yuki Katou,Hirokazu Tanaka,Makiko Komata,Katsuhiko Shirahige,Masashige Bando,Takashi Sutani

doi:10.1074/jbc.m109.065730

Takehiko Itoh, Yuki Katou + Show 5 more

Open Access

https://doi.org/10.1074/jbc.m109.065730

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Abstract

Mrc1 (mediator of replication checkpoint), Tof1 (topoisomerase I interacting factor), and Csm3 (chromosome segregation in meiosis) are checkpoint-mediator proteins that function during DNA replication and activate the effector kinase Rad53. We reported previously that Mrc1 and Tof1 are constituents of the replication machinery and that both proteins are required for the proper arrest and stabilization of replication forks in the presence of hydroxyurea. In our current study, we show that Csm3 is a component of moving replication forks and that both Tof1 and Csm3 are specifically required for the association of Mrc1 with these structures. In contrast, the deletion of mrc1 did not affect the association of Tof1 and Csm3 with the replication fork complex. In agreement with previous observations in yeast cells, the results of a baculovirus coexpression system showed that these three proteins interact directly with each other to form a mediator complex in the absence of replication forks.

Highlights

During the progression of DNA replication, replication forks are exposed to various types of replicative stress
Csm3 Associates with Replication Forks—Previous biochemical studies have revealed that Tof1, Mrc1, and Csm3 are components of the replisome progression complex [11, 25]
Tof1, Csm3, and Mrc1 are essential for activation of the DNA replication checkpoint (8 –10) and for the stabilization of DNA replication forks during pausing under replicative stress, such as that induced by HU treatment [20]

Summary

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EXPERIMENTAL PROCEDURES

RESULTS

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DISCUSSION