Dna modifying enzymes

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).

All restriction enzymes and DNA modifying enzymes were obtained from New England Biolabs (MA, USA). Plasmid pET-30a was obtained from Novagen (Germany). Escherichia coli DH10β and BL21 (DE3) were used as the host for plasmid cloning and protein expression, respectively. Nickel-nitrilotriacetic acid-agarose was purchased from Qiagen (USA). DNA and protein markers were acquired from New England Biolabs, β- NADPH, β-sitosterol and stigmasterol were acquired from Sigma-Aldrich (USA). Other materials used in this study were of analytical grade and were commercially available.