Fission Yeast Swi1-Swi3 Complex Facilitates DNA Binding of Mrc1

Zhiying You,Mika Yokoyama,Taku Tanaka,Hisao Masai,Seiji Matsumoto,Rino Fukatsu

doi:10.1074/jbc.m110.173344

Zhiying You, Mika Yokoyama + Show 4 more

Open Access

https://doi.org/10.1074/jbc.m110.173344

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Abstract

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 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) that 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 that may play an important role in replication pausing and fork stabilization.

Highlights

Strict monitoring of ongoing forks is crucial for genome integrity
In fission yeast, Zhao and Russell [15] have reported that the Nterminal half of the fission yeast Mrc1 binds preferentially to branched DNA in vitro, and the DNA binding domain has been identified. These results suggest that Claspin/Mrc1 directly interacts with various branched structures of DNA associated with fork arrest
It was proposed that Swi1 and Swi3 form a “replication fork protection complex,” which is required for stabilization of stalled forks in fission yeast [16]

Summary

EXPERIMENTAL PROCEDURES

Expression Vectors for Fission Yeast Mrc and Swi1-Swi3— For the expression of Mrc in Escherichia coli cells, full-length Mrc1-FLAG (tagged at the C terminus) was cloned at the BamHI sites of pT7–7/pQE30, resulting in generation of RGS-His6-Mrc1-FLAG. The eluted fraction was mixed with 0.5 ml of anti-FLAG M2 affinity gel (Sigma) equilibrated with buffer B and incubated at 4 °C for 1.5 h on a rotator. After washing three times with 15 ml of buffer B, bound protein was eluted four times by incubation at 4 °C for 20 min each with 0.5 ml of buffer B containing 0.4 mg/ml FLAG peptide. Immunoprecipitation Assay—Immunoprecipitation assays were conducted in 100 ␮l of reaction mixtures containing IP buffer (50 mM Tris-HCl (pH 7.0), 5 mM EDTA, 1% Triton X-100, 1 mM DTT, and 100 mM NaCl) or buffer A (40 mM Hepes/KOH (pH 7.6), 40 mM potassium glutamate, 1 mM EDTA, 1 mM DTT, 8 mM magnesium acetate, 1 mM CaCl2, and 10% glycerol) using IgG purified from anti-Mrc serum. Fission yeast strains used in this study were YM71 (hϪ leu ura4-D18), EN3182 (hϪ leu ura4-D18 swi1::kan), EN3366 (hϪ leu ura4-D18 swi3::kan), Y393 (h90 leu ura4-D18), Y400 (h90 leu ura4-D18 swi1::kan), E111 (h90 his ade210 swi1-111), and JZ277 (h90 his ade6-M210 swi1-E662K)

RESULTS

DISCUSSION

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