ABSTRACT
Activation of the replicative Mcm2-7 helicase by loading GINS and Cdc45 is crucial for replication origin firing, and as such for faithful genetic inheritance. Our biochemical and structural studies demonstrate that the helicase activator GINS interacts with TopBP1 through two separate binding surfaces, the first involving a stretch of highly conserved amino acids in the TopBP1-GINI region, the second a surface on TopBP1-BRCT4. The two surfaces bind to opposite ends of the A domain of the GINS subunit Psf1. Mutation analysis reveals that either surface is individually able to support TopBP1-GINS interaction, albeit with reduced affinity. Consistently, either surface is sufficient for replication origin firing in Xenopus egg extracts and becomes essential in the absence of the other. The TopBP1-GINS interaction appears sterically incompatible with simultaneous binding of DNA polymerase epsilon (Polε) to GINS when bound to Mcm2-7-Cdc45, although TopBP1-BRCT4 and the Polε subunit PolE2 show only partial competitivity in binding to Psf1. Our TopBP1-GINS model improves the understanding of the recently characterised metazoan pre-loading complex. It further predicts the coordination of three molecular origin firing processes, DNA polymerase epsilon arrival, TopBP1 ejection and GINS integration into Mcm2-7-Cdc45.
Subject(s)
DNA Replication , DNA-Binding Proteins , Animals , DNA-Binding Proteins/genetics , DNA-Binding Proteins/metabolism , DNA Polymerase II/metabolism , Cell Cycle Proteins/genetics , Cell Cycle Proteins/metabolism , Minichromosome Maintenance Proteins/metabolism , Virus ReplicationABSTRACT
Faithful genome duplication requires appropriately controlled replication origin firing. The metazoan origin firing regulation hub Treslin/TICRR and its yeast orthologue Sld3 share the Sld3-Treslin domain and the adjacent TopBP1/Dpb11 interaction domain. We report a revised domain architecture model of Treslin/TICRR. Protein sequence analyses uncovered a conserved Ku70-homologous ß-barrel fold in the Treslin/TICRR middle domain (M domain) and in Sld3. Thus, the Sld3-homologous Treslin/TICRR core comprises its three central domains, M domain, Sld3-Treslin domain, and TopBP1/Dpb11 interaction domain, flanked by non-conserved terminal domains, the CIT (conserved in Treslins) and the C terminus. The CIT includes a von Willebrand factor type A domain. Unexpectedly, MTBP, Treslin/TICRR, and Ku70/80 share the same N-terminal domain architecture, von Willebrand factor type A and Ku70-like ß-barrels, suggesting a common ancestry. Binding experiments using mutants and the Sld3-Sld7 dimer structure suggest that the Treslin/Sld3 and MTBP/Sld7 ß-barrels engage in homotypic interactions, reminiscent of Ku70-Ku80 dimerization. Cells expressing Treslin/TICRR domain mutants indicate that all Sld3-core domains and the non-conserved terminal domains fulfil important functions during origin firing in human cells. Thus, metazoa-specific and widely conserved molecular processes cooperate during metazoan origin firing.
Subject(s)
Cell Cycle Proteins/metabolism , Replication Origin/physiology , Carrier Proteins/metabolism , Cell Cycle Proteins/genetics , Cell Cycle Proteins/physiology , Cyclin-Dependent Kinases/metabolism , DNA Replication/genetics , DNA-Binding Proteins/metabolism , Humans , Protein Conformation , Replication Origin/genetics , Structure-Activity RelationshipABSTRACT
Faithful genome duplication requires regulation of origin firing to determine loci, timing and efficiency of replisome generation. Established kinase targets for eukaryotic origin firing regulation are the Mcm2-7 helicase, Sld3/Treslin/TICRR and Sld2/RecQL4. We report that metazoan Sld7, MTBP (Mdm2 binding protein), is targeted by at least three kinase pathways. MTBP was phosphorylated at CDK consensus sites by cell cycle cyclin-dependent kinases (CDK) and Cdk8/19-cyclin C. Phospho-mimetic MTBP CDK site mutants, but not non-phosphorylatable mutants, promoted origin firing in human cells. MTBP was also phosphorylated at DNA damage checkpoint kinase consensus sites. Phospho-mimetic mutations at these sites inhibited MTBP's origin firing capability. Whilst expressing a non-phospho MTBP mutant was insufficient to relieve the suppression of origin firing upon DNA damage, the mutant induced a genome-wide increase of origin firing in unperturbed cells. Our work establishes MTBP as a regulation platform of metazoan origin firing.
Subject(s)
Carrier Proteins/metabolism , DNA Replication , Replication Origin , Animals , Binding Sites , Cell Line , Conserved Sequence , Cyclin-Dependent Kinases/metabolism , DNA Damage , DNA-Binding Proteins/metabolism , Humans , Phosphorylation , Protein Binding , Protein Processing, Post-TranslationalABSTRACT
Accurate genome duplication underlies genetic homeostasis. Metazoan Mdm2 binding protein (MTBP) forms a main regulatory platform for origin firing together with Treslin/TICRR and TopBP1 (Topoisomerase II binding protein 1 (TopBP1)-interacting replication stimulating protein/TopBP1-interacting checkpoint and replication regulator). We report the first comprehensive analysis of MTBP and reveal conserved and metazoa-specific MTBP functions in replication. This suggests that metazoa have evolved specific molecular mechanisms to adapt replication principles conserved with yeast to the specific requirements of the more complex metazoan cells. We uncover one such metazoa-specific process: a new replication factor, cyclin-dependent kinase 8/19-cyclinC (Cdk8/19-cyclin C), binds to a central domain of MTBP. This interaction is required for complete genome duplication in human cells. In the absence of MTBP binding to Cdk8/19-cyclin C, cells enter mitosis with incompletely duplicated chromosomes, and subsequent chromosome segregation occurs inaccurately. Using remote homology searches, we identified MTBP as the metazoan orthologue of yeast synthetic lethal with Dpb11 7 (Sld7). This homology finally demonstrates that the set of yeast core factors sufficient for replication initiation in vitro is conserved in metazoa. MTBP and Sld7 contain two homologous domains that are present in no other protein, one each in the N and C termini. In MTBP the conserved termini flank the metazoa-specific Cdk8/19-cyclin C binding region and are required for normal origin firing in human cells. The N termini of MTBP and Sld7 share an essential origin firing function, the interaction with Treslin/TICRR or its yeast orthologue Sld3, respectively. The C termini may function as homodimerisation domains. Our characterisation of broadly conserved and metazoa-specific initiation processes sets the basis for further mechanistic dissection of replication initiation in vertebrates. It is a first step in understanding the distinctions of origin firing in higher eukaryotes.
