Structural Basis for the Recruitment of Ctf18-RFC to the Replisome

• Published in: Structure (London) 2018-01, Vol.26 (1), p.137-144.e3
• Main Authors: Grabarczyk, Daniel B., Silkenat, Sabrina, Kisker, Caroline
• Format: Article
• Language: English
• Subjects: architecture of the eukaryotic replisome; Binding Sites; Binding, Competitive; Chromosomal Proteins, Non-Histone - chemistry; Chromosomal Proteins, Non-Histone - genetics; Chromosomal Proteins, Non-Histone - metabolism; clamp loader; Cloning, Molecular; cohesion establishment; Crystallography, X-Ray; DNA - chemistry; DNA - genetics; DNA - metabolism; DNA polymerase epsilon; DNA Polymerase II - chemistry; DNA Polymerase II - genetics; DNA Polymerase II - metabolism; DNA Replication; DNA-Binding Proteins - chemistry; DNA-Binding Proteins - genetics; DNA-Binding Proteins - metabolism; Escherichia coli - genetics; Escherichia coli - metabolism; Gene Expression; Genetic Vectors - chemistry; Genetic Vectors - metabolism; Kinetics; Models, Molecular; PCNA; Protein Binding; Protein Conformation, alpha-Helical; Protein Conformation, beta-Strand; Protein Interaction Domains and Motifs; Protein Multimerization; protein-DNA interactions; protein-protein interactions; Recombinant Proteins - chemistry; Recombinant Proteins - genetics; Recombinant Proteins - metabolism; replication fork stalling; Replication Protein C - chemistry; Replication Protein C - genetics; Replication Protein C - metabolism; Saccharomyces cerevisiae - genetics; Saccharomyces cerevisiae - metabolism; Saccharomyces cerevisiae Proteins - chemistry; Saccharomyces cerevisiae Proteins - genetics; Saccharomyces cerevisiae Proteins - metabolism; Substrate Specificity; winged-helix domains
• ISSN: 0969-2126; 1878-4186
• DOI: 10.1016/j.str.2017.11.004

Ctf18-RFC is an alternative PCNA loader which plays important but poorly understood roles in multiple DNA replication-associated processes. To fulfill its specialist roles, the Ctf18-RFC clamp loader contains a unique module in which the Dcc1-Ctf8 complex is bound to the C terminus of Ctf18 (the Ctf18-1-8 module). Here, we report the structural and functional characterization of the heterotetrameric complex formed between Ctf18-1-8 and a 63 kDa fragment of DNA polymerase ɛ. Our data reveal that Ctf18-1-8 binds stably to the polymerase and far from its other functional sites, suggesting that Ctf18-RFC could be associated with Pol ɛ throughout normal replication as the leading strand clamp loader. We also show that Pol ɛ and double-stranded DNA compete to bind the same winged-helix domain on Dcc1, with Pol ɛ being the preferred binding partner, thus suggesting that there are two alternative pathways to recruit Ctf18-RFC to sites of replication. [Display omitted] •Crystal structure of a eukaryotic leading strand polymerase:clamp loader complex•Ctf18-RFC may be associated with the leading strand throughout normal replication•A winged-helix domain on Ctf18-RFC recognizes two distinct substrates•DNA Pol ɛ outcompetes dsDNA for recruiting Ctf18-RFC to the replisome Grabarczyk et al. structurally and functionally characterize the interaction between the yeast leading strand DNA polymerase ɛ and the alternative clamp loader Ctf18-RFC. Their study reveals that Ctf18-RFC may associate with Pol ɛ during normal replication through an unusual bifunctional winged-helix domain.