title: The DNA-Binding Domain of S. pombe Mrc1 (Claspin) Acts to Enhance Stalling at Replication Barriers.
abstract: During S-phase replication forks can stall at specific genetic loci. At some loci, the stalling events depend on the replisome components Schizosaccharomyces pombe Swi1 (Saccharomyces cerevisiae Tof1) and Swi3 (S. cerevisiae Csm3) as well as factors that bind DNA in a site-specific manner. Using a new genetic screen we identified Mrc1 (S. cerevisiae Mrc1/metazoan Claspin) as a replisome component involved in replication stalling. Mrc1 is known to form a sub-complex with Swi1 and Swi3 within the replisome and is required for the intra-S phase checkpoint activation. This discovery is surprising as several studies show that S. cerevisiae Mrc1 is not required for replication barrier activity. In contrast, we show that deletion of S. pombe mrc1 leads to an approximately three-fold reduction in barrier activity at several barriers and that Mrc1's role in replication fork stalling is independent of its role in checkpoint activation. Instead, S. pombe Mrc1 mediated fork stalling requires the presence of a functional copy of its phylogenetically conserved DNA binding domain. Interestingly, this domain is on the sequence level absent from S. cerevisiae Mrc1. Our study indicates that direct interactions between the eukaryotic replisome and the DNA are important for site-specific replication stalling.
authors.affiliation: Warwick Medical School, University of Warwick, Gibbet Hill Campus, CV47AL Coventry, United Kingdom.
authors.forename: Juergen
authors.initials: J
authors.lastname: Zech
authors.affiliation: Warwick Medical School, University of Warwick, Gibbet Hill Campus, CV47AL Coventry, United Kingdom.
authors.forename: Emma Louise
authors.initials: EL
authors.lastname: Godfrey
authors.affiliation: Genome Dynamics Project, Department of Genome Medicine, Tokyo Metropolitan Institute of Medical Science, Setagaya-ku, Tokyo 156-8613, Japan.
authors.forename: Hisao
authors.initials: H
authors.lastname: Masai
authors.affiliation: School of Biological Sciences, Bangor University, Deiniol Road, Bangor, Wales, LI57 2UW, United Kingdom.
authors.forename: Edgar
authors.initials: E
authors.lastname: Hartsuiker
authors.affiliation: Warwick Medical School, University of Warwick, Gibbet Hill Campus, CV47AL Coventry, United Kingdom.
authors.forename: Jacob Zeuthen
authors.initials: JZ
authors.lastname: Dalgaard
mesh_terms: D001665:Binding Sites; D018797:Cell Cycle Proteins / Q000378:metabolism; D004261:DNA Replication; D004271:DNA, Fungal / Q000378:metabolism*; D004268:DNA-Binding Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D005820:Genetic Testing / Q000379:methods; D016196:S Phase; D012568:Schizosaccharomyces / Q000235:genetics* / Q000378:metabolism; D029702:Schizosaccharomyces pombe Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*
chemical_list: D018797:Cell Cycle Proteins; D004271:DNA, Fungal; D004268:DNA-Binding Proteins; C533276:MRC1 protein, S pombe; D029702:Schizosaccharomyces pombe Proteins; C499635:Swi3 protein, S pombe; C513096:swi1 protein, S pombe