Structural Insights into Pol-β Repair of O6MeG Lesions

Polβ was expressed and purified
as described previously.^{35 (link)} Polβ binary
complex with a single-nucleotide
gap opposite templating O6MeG was prepared using the same conditions
described previously.^{35 (link)} Polβ ternary
complex was prepared by adding nonhydrolyzable dCMPNPP or dTMPNPP
(5.0 mM, Jena Biosciences) to the mixture of the polβ gapped
binary complex. Polβ ternary complex crystals with nonhydrolyzable
dCMPNPP or dTMPNPP opposite templating O6MeG were grown over 2–4
weeks in a buffer solution containing 50 mM imidazole, pH 7.5, 14%–23%
PEG3400, and 350 mM NaOAc.^{35 (link)} The polβ
binary and ternary complex crystals were cryo-protected with 12% ethylene
glycol and flash-frozen in liquid nitrogen. Diffraction data were
collected at the beamline 5.0.3 at the Advanced Light Source, Lawrence
Berkeley National Laboratory and were processed using the HK-2000
program. The polβ gapped binary complex structure and the ternary
complex structures were solved by molecular replacement^{36 (link)} using published binary (PDB ID 1BPX) and ternary (PDB
ID 1BPY) structures
as the search models, respectively.^{37 (link)} The
model building and structure refinement were conducted using COOT,^{38 (link)} Phenix,^{39 (link)} and MolProbity,^{40 (link)} and all the crystallographic figures were generated
using PyMOL.