Computational protocol: Mechanism of error-free replication across benzo[a]pyrene stereoisomers by Rev1 DNA polymerase

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Protocol publication

[…] The X-ray diffraction data were collected at the NSLSX25 beam line at the Brookhaven National Laboratory. The data were processed and scaled using the HKL2000 suite. The structure of the 10R ( + )-cis-BP-dG complex was solved by the molecular replacement method (Phaser) in the CCP4 program package, using a previously published structure with an unmodified dG and incoming dCTP, PDB ID 2AQ4 (with a different DNA sequence) as search model. The model building, including substitution of the DNA sequence, was finished manually in Coot based on the electron density maps calculated in REFMAC in the CCP4 suite. The resulting model was refined in REFMAC in space group P 21212 with a = 63 Å, b = 180 Å, c = 55 Å unit cell to 1.92 Å and Rwork/free 18.3%/21.8%, correspondently. The placement of the BP and modified dG moieties was verified using the simulated annealing omit maps calculated in Phenix with the (+)-cis-BP-N 2-dG omitted from the models before heating them to 2000 K and then slowly cooling them. The refined 10R (+)-cis-BP-dG structure includes Rev1 residues 307–738, nucleotides 2–17 for the template strand, nucleotides 1–12 for the primer strand, incoming dCTP, 4 Mg2+ ions, and 311 water molecules. The 3′-terminal and the adjacent residue of the primer strand have double conformations of their phosphate backbone reflecting the mobility of the 3′ terminus in Rev1 complex. The structure of 10S (+)-trans-BP-dG ternary complex was obtained by molecular replacement using the 10R (+)-cis-BP complex as a search model. The placement of the 10S (+)-trans-BP- N 2-dG adduct was verified using the simulated annealing omit maps as described above. The model was refined to 1.85 Å in space group P 212121; a = 64 Å, b = 65 Å, c = 131 Å and Rwork/free 17.7%/21.7%, respectively. […]

Pipeline specifications

Software tools CCP4, Coot, PHENIX
Databases CIS-BP
Applications Small-angle scattering, Protein structure analysis
Organisms Saccharomyces cerevisiae
Chemicals Benzo(a)pyrene, Guanine