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9 protocols using dna modification enzymes

1

Radioactive Nucleotide Biochemical Assays

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Radioactive nucleotides were from PerkinElmer Life Sciences (Waltham, Massachusetts). Unlabeled ATP was from Cytiva (Marlborough, MA). ATPγS was from Roche (Basel, Switzerland). DNA-modification enzymes were from New England BioLabs (Ipswich.Massachusetts). DNA oligonucleotides were from Integrated DNA Technologies (Coralville, Iowa). Protein concentrations were determined with the Bio-Rad Labs (Hercules, California) Bradford Protein stain using bovine serum albumin as a standard. Streptavidin-coated Dynabead M-280 magnetic beads were purchased from Thermo-Fisher Scientific (Waltham, Massachusetts). Anti-Digoxigenin, the anti-Dig Fab, was from Millipore-Sigma (St. Louis, MO).
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2

Purification and Characterization of CMG and Mcm10

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Radioactive nucleotides were from Perkin Elmer and unlabeled nucleotides were from GE Healthcare. DNA modification enzymes were from New England Biolabs. DNA oligonucleotides were from Integrated DNA Technologies except for those with methylphosphonate linkages which were from Biosynthesis (Lewisville, TX). CMG and Mcm10 were overexpressed and purified as previously described (Georgescu et al., 2014 (link); Langston et al., 2017 (link); Langston et al., 2014 (link)). Protein concentrations were determined using the Bio-Rad Bradford Protein stain using BSA as a standard.
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3

Molecular Cloning Toolkit for Research

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KOD FX Neo DNA polymerase was purchased from Toyobo (Osaka, Japan). EmeraldAmp PCR Master Mix and In-Fusion® Cloning Kit were purchased from Takara Bio (Kusatsu, Japan). Restriction enzymes and DNA modification enzymes were purchased from New England Biolabs (Ipswich, MA, USA). Oligonucleotides were purchased from Thermo Fisher Scientific (Waltham, MA, USA). 5-Fluoroorotate (5-FOA) was purchased from Sigma-Aldrich (St. Louis, MO, USA). Agar, ampicillin (Amp), and hygromycin B (Hg) were purchased from Wako Pure Chemical/FUJIFILM (Osaka, Japan). Lennox LB [1% (w/v) tryptone, 0.5% (w/v) yeast extract, 1% (w/v) NaCl] was purchased from Nacalai (Kyoto, Japan).
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4

Biochemical Characterization of T-Antigen

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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-Ag131–627, 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.
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5

Protein Purification and Characterization

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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.
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6

Protein purification using buffers

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Radioactive nucleotides were from PerkinElmer. Unlabeled nucleotides were from GE Healthcare. DNA-modification enzymes were from New England BioLabs. DNA oligonucleotides were from Integrated DNA Technologies. Protein concentrations were determined with 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 mM 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 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. Buffer D is 25 mM Tris-OAc, pH 7.6, 40 mM KOAc, 40 mM K glutamate, 2 mM MgOAc2, 1 mM DTT, 20% glycerol and 0.25 mM EDTA. Stop buffer is 1% SDS, 40 mm EDTA.
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7

Plasmid Transformation in E. coli and S. aureus

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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.
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8

Isolation and Purification of Bacterial DNA

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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).
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9

Replication Protein Complex Purification

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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.
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