Mechanisms in helicase activation and analysis of the replication fork activity and architecture

Background on pre-initiation complex (pre-IC) assembly
Pre-RC formation requires the absence of CDK activity in G1. Once the pre-RC is formed, S-CDK and DDK activity trigger origin firing. At the same time CDK activity destroys origin competence and in consequence new pre-RCs cannot form anymore on DNA. This kinase-dependent switch guarantees that each origin fires only once and is required for genomic stability. At the same time, kinase activity promotes formation of a pre-initiation complex (pre-IC) and eventually DNA synthesis. The assembly of the pre-IC is illustrated in the schematic diagram on the right. Click on the diagram to enlarge it. The essential targets of S-CDK are Sld2 and Sld3. Phosphorylation of Sld2 and Sld3 promotes their binding to Dbp11, which is required for the loading of the DNA polymerase onto origin DNA. MCM’s are likely the essential target of DDK, since a Mcm5 mutant, bob1, bypasses the requirement for DDK activity. DDK phosphorylates specifically loaded MCM2-7 and results in a structural change of unknown function. Moreover DDK activity is required for Cdc45 and GINS binding to the pre-IC. A consequence of CDK and DDK activity is that RPA binds to the origin, which implies that ss-DNA is generated during pre-IC formation. It is believed that the DNA helicase is involved in generation of ss-DNA, since an MCM ATPase mutant fails to efficiently attract RPA to chromatin. The exact functions of Cdc45, Sld3, GINS and most of the other pre-IC proteins are not known.

Pre-IC assembly
Figure 1. Pre-IC assembly