Tanaka T, Yokoyama M, Matsumoto S, Fukatsu R, You Z, Masai H

Replication fork protection complex Swi1-Swi3 and replication checkpoint mediator Mrc1 are required for maintenance of replication fork integrity during the course of DNA replication in the fission yeast Schizosaccharomyces pombe. These proteins play crucial roles in stabilizing stalled forks and activating replication checkpoint signaling pathways. Although they are conserved replication ...

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fork components, precise biochemical roles of these proteins are not known. Here we purified Mrc1 and Swi1-Swi3 proteins, and show that these proteins bind to DNA independently but synergistically in vitro. Mrc1 binds preferentially to arrested-fork or D-loop-like structures although the affinity is relatively low, whereas the Swi1-Swi3 complex binds to double-stranded DNA with higher affinity. In the presence of a low concentration of Swi1-Swi3, Mrc1 generates a novel ternary complex and binds to various types of DNA with higher affinity. Moreover, purified Mrc1 and Swi1-Swi3 physically interact with each other, and this interaction is lost by mutations in the known DNA binding domain of Mrc1 (K235E, K236E). The interaction is also lost in a mutant form of Swi1 (E662K), which is specifically defective in polar fork arrest at a site called RTS1 and causes sensitivity to genotoxic agents, although the DNA binding affinity of Swi1-Swi3 is not affected by this mutation. As expected, the synergistic effect of the Swi1-Swi3 on DNA binding of Mrc1 is also lost by these mutations affecting the interaction between Mrc1 and Swi1-Swi3. Our results reveal an aspect of molecular interactions which may play an important role in replication pausing and fork stabilization.

Date: Oct. 05, 2010

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