ABSTRACT
Sister-chromatid cohesion is established by Eco1-mediated acetylation on two conserved tandem lysines in the cohesin Smc3 subunit. However, the molecular basis of Eco1 substrate recognition and acetylation in cohesion is not fully understood. Here, we discover and rationalize the substrate specificity of Eco1 using mass spectrometry coupled with in-vitro acetylation assays and crystallography. Our structures of the X. laevis Eco2 (xEco2) bound to its primary and secondary Smc3 substrates demonstrate the plasticity of the substrate-binding site, which confers substrate specificity by concerted conformational changes of the central ß hairpin and the C-terminal extension.
Subject(s)
Acetyltransferases/chemistry , Cell Cycle Proteins/chemistry , Chromosomal Proteins, Non-Histone/chemistry , Chromosome Segregation , Nuclear Proteins/chemistry , Saccharomyces cerevisiae Proteins/chemistry , Xenopus Proteins/chemistry , Acetylation , Acetyltransferases/genetics , Acetyltransferases/metabolism , Amino Acid Sequence , Animals , Binding Sites , Cell Cycle Proteins/genetics , Cell Cycle Proteins/metabolism , Chromosomal Proteins, Non-Histone/genetics , Chromosomal Proteins, Non-Histone/metabolism , Crystallography, X-Ray , Gene Expression , Models, Molecular , Nuclear Proteins/genetics , Nuclear Proteins/metabolism , Protein Binding , Protein Conformation, alpha-Helical , Protein Conformation, beta-Strand , Protein Interaction Domains and Motifs , Recombinant Proteins/chemistry , Recombinant Proteins/genetics , Recombinant Proteins/metabolism , Saccharomyces cerevisiae/genetics , Saccharomyces cerevisiae/metabolism , Saccharomyces cerevisiae/ultrastructure , Saccharomyces cerevisiae Proteins/genetics , Saccharomyces cerevisiae Proteins/metabolism , Sequence Alignment , Sequence Homology, Amino Acid , Substrate Specificity , Xenopus Proteins/genetics , Xenopus Proteins/metabolism , Xenopus laevis/genetics , Xenopus laevis/metabolismABSTRACT
Replication factor C complexes load and unload processivity clamps from DNA and are involved in multiple DNA replication and repair pathways. The RFCCtf18 variant complex is required for activation of the intra-S-phase checkpoint at stalled replication forks and aids the establishment of sister chromatid cohesion. Unlike other RFC complexes, RFCCtf18 contains two non-Rfc subunits, Dcc1 and Ctf8. Here, we present the crystal structure of the Dcc1-Ctf8 heterodimer bound to the C-terminus of Ctf18. We find that the C-terminus of Dcc1 contains three-winged helix domains, which bind to both ssDNA and dsDNA We further show that these domains are required for full recruitment of the complex to chromatin, and correct activation of the replication checkpoint. These findings provide the first structural data on a eukaryotic seven-subunit clamp loader and define a new biochemical activity for Dcc1.
Subject(s)
Chromatin/metabolism , DNA-Binding Proteins/chemistry , Models, Molecular , Protein Conformation , Saccharomyces cerevisiae Proteins/chemistry , Amino Acid Sequence , DNA-Binding Proteins/metabolism , Protein Binding , Protein Interaction Domains and Motifs , Protein Multimerization , Saccharomyces cerevisiae Proteins/metabolism , Structure-Activity RelationshipABSTRACT
The functions of cohesin are central to genome integrity, chromosome organization and transcription regulation through its prevention of premature sister-chromatid separation and the formation of DNA loops. The loading of cohesin onto chromatin depends on the Scc2-Scc4 complex; however, little is known about how it stimulates the cohesion-loading activity. Here we determine the large 'hook' structure of Scc2 responsible for catalysing cohesin loading. We identify key Scc2 surfaces that are crucial for cohesin loading in vivo. With the aid of previously determined structures and homology modelling, we derive a pseudo-atomic structure of the full-length Scc2-Scc4 complex. Finally, using recombinantly purified Scc2-Scc4 and cohesin, we performed crosslinking mass spectrometry and interaction assays that suggest Scc2-Scc4 uses its modular structure to make multiple contacts with cohesin.