Fluorescent Labeling of Replication Proteins

Wild-type ORC and Cdc6 were purified as described previously (Frigola et al., 2013 (link)). We used a N- and C-terminal protein modifications to fluorescently label ORC (-LPETGG at the C-terminus of Orc5 and Orc6, Ubiquitin-GGG-Flag at the N-terminus of Orc1), Mcm2-7 (LPETGG at the C-terminus of Mcm2, Ubiquitin-GGG-Flag at the N-terminus of Mcm4, 3xFlag-TEV[ENLYFQ/G]-GG at the N-terminus of Mcm3), and Cdt1 (Ubiquitin-GGG tag at the N-terminus, LPETGG tag at the C-terminus). The ubiquitin (in vivo) and 3xFlag-TEV (using TEV protease, NEB) fusions were removed to reveal three N-terminal glycines required for N-terminal sortase-mediated labeling. The peptides NH₂-CHHHHHHHHHLPETGG-COOH and NH₂-GGGHHHHHHHHHHC-COOH were used for N- and C-terminal labeling, respectively, and will be referred to as the N-peptide and C-peptide. The N- and C-peptides were labeled with maleimide-derivatized DY549P1 (Dyomics), DY649P1 (Dyomics), Dylight550 (Thermo-scientific), Dylight650 (Thermo-scientific), or Cy3B (Cytiva) via cystine–maleimide conjugation as described previously (Ticau et al., 2015 (link)). Sortase was used to couple the fluorescently labeled peptide to the N- or C-terminus of the helicase-loading proteins as described below. The peptide-coupled proteins were separated from uncoupled proteins using Ni-NTA Agarose (Qiagen).