PCNA Loading and Unloading Assays

Radioactive nucleotides were from PerkinElmer Life Sciences (Waltham, MA). Unlabeled ATP was from Cytiva (Marlborough, MA). ATPγS and AMP-PNP were from Roche (Basel, Switzerland). Apyrase from potato was from Sigma-Aldrich (St. Louis, MO). DNA-modifying enzymes were from New England Biolabs (NEB; Ipswich, MA). Protein concentrations were determined with the Bradford Protein stain (Bio-Rad Labs, Hercules, CA) using bovine serum albumin (BSA) as a standard. PCNA containing a hexahistidine tag and a six-residue site for the catalytic subunit of cyclic adenosine 3′,5′-monophosphate (cAMP)–dependent protein kinase A at the N terminus was cloned, expressed, purified, and radiolabeled with ³²P-ATP as described earlier (8 (link), 59 (link)). For ³²P-PCNA loading, we used the yeast RFC lacking the N-terminal residues 3 to 273 as described (60 (link)). Full-length RFC was used for ³²P-PCNA unloading experiments (61 (link)). RPA was purified as described (62 (link)). S.c. Pol d was purified as described (63 (link)). ϕX174 ssDNA was from Roche (Basel, Switzerland). The 18 30-mer DNA oligos that hybridize nearly equally around the ssDNA of ϕX174 ssDNA were from IDT (see table S2).