Publication protocol
"Plasmids with and without DNA damage were prepared as described previously [3] by insertion of oligonucleotides into cassettes at specific sites either ~ 3 kb (plasmid ZN3) or 4.5 kb (plasmid ZN5Sp) from the origin using the following oligonucleotides: undamaged DNA: 5′-phos-TCAGCACTTAAGTCC; THF: 5′-phos-TCAGCACT-/idSp/-AAGTCC, CPD: 5′-phos-TCAGCAC-/CPD/-AAGTCC. To generate replication assay templates, plasmids were all linearized with AhdI as previously described [3], and in the following cases additionally with AsiSI (Fig. 3 and Supplemental Fig. S3) and PsiI (Fig. 4 and Supplemental Fig. S4). The AB template was prepared by inserting an oligonucleotide containing deoxyuracil (5′phos-TCGCACT/ideoxyU/AAGTCC), and after linearization with AhdI, the template was treated with UDG (NEB M0280) in the same buffer for 1 h. In Fig. 4, the undamaged template was prepared by linearizing Maxi prep DNA.
Standard replication assays were performed exactly as described in detail previously [3]. Briefly, MCM2–7 loading was performed at 24 °C for 10 min, and S-CDK was added for a further 5 min before the reaction was initiated and transferred to 30 °C. The final reaction buffer composition (except where indicated) was as follows: 29.2 mM Hepes–KOH (pH 7.6), 217 mM potassium glutamate (except Fig. 3, Fig. 4d; Supplemental Figs. S3, S4F: 117 mM), 0.0117% NP-40-S, 1.17 mM DTT, 11.7 mM Mg(OAc)2, 0.117 mg/ml BSA, 6.7 mM KCl, 3 mM ATP, 400 μM CTP, GTP, UTP, 30 μM dATP, dCTP, dGTP, dTTP, 33 nM α-[32P]-dCTP, 12.5 nM Cdt1/Mcm2–7, 7.5 nM Cdc6, 3.3 nM ORC, 8.3 nM DDK, 20 nM S-CDK, 30 nM Dpb11, 210 nM GINS, 40 nM Cdc45, 20 nM Pol ε, 5 nM Mcm10, 20 nM Ctf4, 60 nM RPA, 20 nM Csm3/Tof1, 20 nM Mrc1, 20 nM RFC, 20 nM PCNA, 10 nM TopoI, 20 nM Pol α, 5 nM Pol δ, 25 nM Sld3/7, and 50 nM Sld2. In pulse-chase experiments in Fig. 3 and Supplemental Fig. S3, Pol δ was omitted, and the reaction was initiated at a lower dCTP concentration (2.5 μM), and after 2 min 50 s, the concentration of all four dNTPs increased to 400 μM. Reactions were quenched with 25–30 mM EDTA except in Fig. 2 and Supplemental Fig. S2, where after 90 min the reaction mix was diluted 4-fold in 25 mM Hepes–KOH (pH 7.6), 10 mM Mg(OAc)2, and 60 nM RPA before the addition of water (negative control), PmeI, AvrII, or AfeI (NEB) (3 μl enzyme in 56 μl diluted reaction mix) and incubation at 37 °C for 10 min, followed by quenching with EDTA.
Following quenching, proteins were removed with 0.1% SDS and 1/100 volumes proteinase K (NEB P8107) at 37 °C for 20 min, and the DNA was extracted with phenol–chloroform. Samples were passed over illustra MicroSpin G-50 columns (GE Healthcare). Where indicated in figures, samples for the denaturing gel were digested with SmaI. Samples were resolved in native and alkaline (denaturing) agarose gels and visualized by autoradiography or phosphorimaging as described in detail previously [3]."
Full paper
Login or
join for free to view the full paper.