Cdc45-ts mutants are defective for DNA replication.

(A) DNA replication products produced with the indicated Cdc45 proteins were separated on a 0.8% alkaline agarose gel and imaged using a phosphoimager. Relative intensities of +DDK lanes were quantified and plotted using ImageJ (Cdc45 = red, Cdc45-124 = blue, Cdc45-238 = green, Cdc45-485 = purple). Horizontal lines indicate the most highly represented product length for each Cdc45 protein tested. (B) Relative levels of DNA replication for the indicated Cdc45 proteins from six experimental replicates of replication assays performed with the indicated Cdc45 mutant proteins were quantified and plotted. (C) Proteins associated with the DNA at the end of the replication reaction. Bead-associated proteins were washed with Buffer H + 0.3M K-Glut 0.02%NP-40, released with DNase, and detected by immunoblot. (D) Relative association of Cdc45, Mcm2-7, GINS and Pol ε with origin DNA after replication. Six (Cdc45 and GINS association) and five (Pol ε association) experimental replicates were quantified and plotted. For both (B) and (D), error bars represent standard error from the mean. Asterisks indicate the following p-values: p≤0.01(**), p≤0.001(***), p≤0.0001(****), not significant (n.s., p≥0.05).