Helicase activation and establishment of replication forks at chromosomal origins of replication

Abstract

Many replication proteins assemble on the pre-RC-formed replication origins and constitute the pre-initiation complex (pre-IC). This complex formation facilitates the conversion of Mcm2-7 in the pre-RC to an active DNA helicase, the Cdc45-Mcm-GINS (CMG) complex. Two protein kinases, cyclin-dependent kinase (CDK) and Dbf4-dependent kinase (DDK), work to complete the formation of the pre-IC. Each kinase is responsible for a distinct step of the process in yeast; Cdc45 associates with origins in a DDK-dependent manner, whereas the association of GINS with origins depends on CDK. These associations with origins also require specific initiation proteins: Sld3 for Cdc45; and Dpb11, Sld2, and Sld3 for GINS. Functional homologs of these proteins exist in metazoa, although pre-IC formation cannot be separated by requirement of DDK and CDK because of experimental limitations. Once the replicative helicase is activated, the origin DNA is unwound, and bidirectional replication forks are established.

Figure 1.

Schematic drawings of the formation of the pre-IC and the initiation of DNA replication. See text for details.

Figure 2.

Schematic drawings of the structure of budding yeast Mcm2, Mcm4, and Mcm6. (Orange) Unstructured amino-terminal portion, (light green) conserved OB-fold like domain, (light blue) conserved AAA⁺-type ATPase domain. The numbers on top indicate the positions of amino acid residues. (White arrow) The amino-terminal portion of Mcm4 corresponding to the Δ74–174 mutation. The positions of alanine substitutions in the phosphorylation site mutants (Randell et al. 2010) are shown by arrowheads; (green) AD/E + ASP/Q, (blue) AP + AQ.

Figure 3.

Schematic drawings of Dpb11/TopBP1 and Sld3/Treslin/Ticrr proteins. (A) Dpb11/TopBP1 proteins. (Light blue) BRCT domains. (B) Sld3/Treslin/Ticrr proteins. (Magenta) The Sld3/Treslin domain; (red) the conserved short patch that contains conserved CDK phosphorylation sites; (orange) the amino-terminal region that is conserved in animal and plant Treslin. (C) Multiple alignments of amino acid sequences that contain conserved CDK phosphorylation sites. (Red asterisks in the top row) Conserved serine and threonine.