A reconstituted system reveals how activating and inhibitory interactions control DDK dependent assembly of the eukaryotic replicative helicase.
During G1-phase of the cell-cycle the replicative MCM2-7 helicase becomes loaded onto DNA into pre-replicative complexes (pre-RCs), resulting in MCM2-7 double-hexamers on DNA. In S-phase, Dbf4-dependent kinase (DDK) and cyclin-dependent-kinase (CDK) direct with the help of a large number of helicase-activation factors the assembly of a Cdc45-MCM2-7-GINS (CMG) complex. However, in the absence of S-phase kinases complex assembly is inhibited, which is unexpected, as the MCM2-7 double-hexamer represents a very large interaction surface. Currently it is unclear what mechanisms restricts complex assembly and how DDK can overcome this inhibition to promote CMG-assembly. We developed an advanced reconstituted-system to study helicase activation in-solution and discovered that individual factors like Sld3 and Sld2 can bind directly to the pre-RC, while Cdc45 cannot. When Sld3 and Sld2 were incubated together with the pre-RC, we observed that competitive interactions restrict complex assembly. DDK stabilizes the Sld3/Sld2-pre-RC complex, but the complex is only short-lived, indicating an anti-cooperative mechanism. Yet, a Sld3/Cdc45-pre-RC can form in the presence of DDK and the addition of Sld2 enhances complex stability. Our results indicate that helicase activation is regulated by competitive and cooperative interactions, which restrict illegitimate complex formation and direct limiting helicase-activation factors into pre-initiation complexes.
DNA Replication Group, Institute of Clinical Sciences, Imperial College London, London W12 0NN, UK.
M Carmen
MC
Herrera
DNA Replication Group, Institute of Clinical Sciences, Imperial College London, London W12 0NN, UK.
Silvia
S
Tognetti
DNA Replication Group, Institute of Clinical Sciences, Imperial College London, London W12 0NN, UK.
Alberto
A
Riera
DNA Replication Group, Institute of Clinical Sciences, Imperial College London, London W12 0NN, UK.
Juergen
J
Zech
DNA Replication Group, Institute of Clinical Sciences, Imperial College London, London W12 0NN, UK.
Pippa
P
Clarke
DNA Replication Group, Institute of Clinical Sciences, Imperial College London, London W12 0NN, UK.
Alejandra
A
Fernández-Cid
DNA Replication Group, Institute of Clinical Sciences, Imperial College London, London W12 0NN, UK chris.speck@imperial.ac.uk.
Christian
http://orcid.org/0000-0001-6646-1692
C
Speck
D002352:Carrier Proteins / Q000378:metabolism; D018797:Cell Cycle Proteins / Q000378:metabolism*; D004271:DNA, Fungal / Q000378:metabolism; D004268:DNA-Binding Proteins / Q000378:metabolism; D064110:Minichromosome Maintenance Proteins / Q000737:chemistry / Q000378:metabolism*; D009687:Nuclear Proteins / Q000378:metabolism; D017346:Protein Serine-Threonine Kinases / Q000378:metabolism*; D018741:Replication Origin; D029701:Saccharomyces cerevisiae Proteins / Q000378:metabolism*
C102209:CDC45 protein, S cerevisiae; D002352:Carrier Proteins; D018797:Cell Cycle Proteins; D004271:DNA, Fungal; D004268:DNA-Binding Proteins; C097185:DPB11 protein, S cerevisiae; D009687:Nuclear Proteins; C115043:SLD2 protein, S cerevisiae; D029701:Saccharomyces cerevisiae Proteins; C426020:Sld3 protein, S cerevisiae; C559742:Sld7 protein, S cerevisiae; C489404:CDC7 protein, S cerevisiae; D017346:Protein Serine-Threonine Kinases; D064110:Minichromosome Maintenance Proteins