Dna modification enzymes

Radioactive nucleotides were from Perkin Elmer. ATP was from Cytiva and adenylyl imidodiphosphate (AMP-PNP) was from Roche. DNA modification enzymes were from New England Biolabs. DNA oligonucleotides were from Integrated DNA Technologies except for those with MeP linkages, which were from Gene Link (Elmsford, NY). Full-length T-Antigen was from Millipore Sigma (SRP2093), and the N-terminal truncation, T-Ag^131–627, of T-Ag was expressed in E. coli as detailed in the SI Appendix. Protein concentrations were determined using the Bio-Rad Bradford Protein stain using BSA as a standard.

Radioactive nucleotides were from Perkin Elmer. Unlabeled nucleotides were from GE Healthcare. DNA modification enzymes and ϕ29 DNA polymerase were from New England Biolabs. DNA oligonucleotides were from Integrated DNA Technologies. Protein concentrations were determined using the Bio-Rad Bradford Protein stain and bovine serum albumin as a standard. Buffer A is 20 mM Tris-HCl, pH 7.5, 5 mM DTT, 0.1 mM EDTA, and 4% glycerol. Buffer B is the same as buffer A except 20 mM Tris-acetate, pH 7.5 was used in place of 20 nM Tris-HCl. Buffer C is 25 mM Tris-Cl pH 7.9, 10% glycerol, 1 mM DTT, 1 mM MgCl₂, 5 mM imidazole, 20 mM KOAc, and 350 mM KCl. Buffer D is 25 mM Tris-OAc pH 7.6, 40 mM K-OAc, 40 mM K glutamate, 2 mM Mg-OAc₂, 1 mM DTT, 20% glycerol, and 0.25 mM EDTA. Stop buffer is 1% SDS, 40 mm EDTA. Buffer H is 20 mM Hepes pH 7.5, 10% glycerol, 1 mM EDTA, 2 mM DTT, 350 mM KCl, 1 mM ATP, and 4 mM MgCl₂.

The restriction enzymes and DNA modification enzymes (New England Biolabs) were used for plasmid construction. The plasmid miniprep kit (Omega) was used for plasmid extraction according to the manufacturer’s instructions. Plasmid DNA was transformed E. coli by the method of Hanahan and Meselson [17 (link)] and electroporated into S. aureus RN4220 with a gene pulser (Bio-Rad). The plasmids from RN4220 were introduced into target strains of S. aureus by transduction with ϕ 85.

DNA and plasmid isolation were performed using standard methods (Ausubel et al., 2002 ). Restriction endonucleases and DNA modification enzymes were purchased from New England Biolabs. Chromosomal DNA from all bacteria was isolated using DNeasy tissue kits (Qiagen); plasmid isolations were performed using QIAprep spin miniprep kits (Qiagen). For both kits, S. aureus cells were lysed by adding 25 μg of lysostaphin (Sigma–Aldrich) to the kit lysis buffer and incubating at 37°C for 30 m prior to following the manufacturer’s protocol. DNA fragments were purified using QIAquick mini-elute PCR purification kits, and PCR was performed using GoTaq Green (Promega). Primers used in this study are indicated in Supplementary Table S3. DNA sequencing was performed by automated sequencing technology through Macrogen Co. (Macrogen USA).

Radioactive nucleotides were from Perkin Elmer, and unlabeled nucleotides were from GE Healthcare. DNA modification enzymes were from New England Biolabs. CMG and Mcm10 were overexpressed and purified as previously described (33 (link), 49 (link), 50 (link)). Protein concentrations were determined using the Bio-Rad Bradford Protein stain using BSA as a standard. DNA oligonucleotides were from Integrated DNA Technologies except for those with Mep linkages which were from Biosynthesis or Gene Link.