Structure-Function Relationships in Miscoding by Sulfolobus solfataricus DNA Polymerase Dpo4: GUANINE N2,N2-DIMETHYL SUBSTITUTION PRODUCES INACTIVE AND MISCODING POLYMERASE COMPLEXES

Kinetic analysis of bypass of abasic site by the catalytic core of yeast DNA polymerase eta

“…An Acquity UPLC BEH octadecylsilane (C 18 ) column (1.7 m, 1.0 mm × 100 mm) was used with the following LC conditions (all at 50 • C) with Buffer A (10 mM NH 4 CH 3 CO 2 plus 2% CH 3 CN (v/v)) and Buffer B (10 mM NH 4 CH 3 CO 2 plus 95% CH 3 CN (v/v)). The conditions used were similar to those previously reported [25,26]. The calculations of the CID fragmentations of oligonucleotide sequences were done using a program linked to the mass spectrometry group (Medicinal Chemistry) of the University of Utah (www.medlib.med.utah.edu/massspec).…”

“…Escherichia coli uracil DNA glycosylase (20 units, Sigma-Aldrich, St. Louis, MO) was added; and the solution was incubated at 37 • C for 6 h to remove the uracil residues on the extended primer and then heated at 95 • C for 1 h in the presence of 0.5 M piperidine, followed by removal of the solvent by in vacuum centrifugation [25,26]. The dried sample was resuspended in 100 l of H 2 O for MS analysis.…”

“…After removal of excess ATP by Bio-Spin 6 column (Bio-Rad, Hercules, CA), the labeled primer and the template (molar ratio 1:1.2) were heated at 95 • C for 5 min and then slowly cooled to room temperature to form the 13-mer/18-mer or 14-mer/18-mer primer/template duplexes, which was used for steady-state and pre-steady-state kinetic experiments [25]. The 13-mer primer containing uracil and 18-mer templates containing G or 8-oxodG (molar ratio 1:1.2) were heated at 95 • C for 5 min and then slowly cooled to room temperature to form the 13-mer/18-mer primer/template duplexes used for LC-MS/MS sequence analysis [25,26].…”

“…After reaction, 5-l aliquots were quenched by the addition of EDTA-formamide solution (50 l of 20 mM EDTA in 95% formamide (v/v) with 0.5% bromphenol blue (w/v) and 0.05% xylene cyanol (w/v)). Products were resolved using 20% polyacrylamide (w/v) denaturing gel electrophoresis (with 8 M urea), visualized and quantitated by phosphor imaging analysis using a Bio-Rad Molecular Imager FX instrument and Quantity One software [26].…”

Structural Mechanism of Ribonucleotide Discrimination by a Y-Family DNA Polymerase

“…11,[20][21][22] The Y-family polymerases have evolved open, solvent-accessible active sites, which accommodate bulky and distorting DNA lesions. [23][24][25][26][27][28][29][30] Consequently, these polymerases can accommodate an incoming nucleotide in different conformations, which allows permissive basepairing to facilitate translesion DNA synthesis. Remarkably, the solvent-accessible active sites of the Y-family polymerases, which have minimal contacts to incoming nucleotides, are still highly discriminatory against NTPs.…”