(A) Structure of the Pyrobaculum aerophilum Cdc6 orthologue with bound ADP (Liu et al., 2000) highlighting the ATP binding pocket and key residues. The ‘DD’ Walker B residues are colored red and the sensor I residue is colored orange. (B) Multiple sequence alignment of Cdc6 Walker B motif. Uppercase letters in consensus are highly conserved. From top to bottom: Homo sapiens, Xenopus laevis, S. pombe, S. cerevisiae, Mus musculus, Drosophila melanogaster, C. elegans, and Pyrobaculum aerophilum Cdc6 homologues. (C) Viability analysis of Cdc6 Walker B motif mutants reveals only E224 is essential. M4466 (cdc6Δ::ura3 pRS416-CDC6) was transformed with the indicated pMW71-derived plasmids (listed in Supplementary file 2) and then restreaked onto SCM-Leu plates representing growth of CDC6/cdc6 or FOA plates, growth of cdc6 mutant only. (D) Growth of plasmid cdc6 derivatives transformed into wild-type yeast (W303-1A) reveals that multiple E224 substitutions have dominant growth affects. (E) Summary of Cdc6 mutational analysis. Shaded regions are non-viable mutants. (F) Flow cytometry profiles of strains M4759 (K4055 × pRS415, vector), M4758 (K4055 × pMW71, CDC6-WT), M4760 (K4055 × pFJ21, cdc6-EQ), and M4762 (K4055 × pFJ230, cdc6-NQ) after addition of methionine to repress expression of wild type MET3p-CDC6 present in all strains.