Journal: Molecular Microbiology
Article Title: Escherichia coli DNA polymerase III is responsible for the high level of spontaneous mutations in mutT strains
Figure Lengend Snippet: Standing start extension of primers having 8-oxo-G at the termini by pol III* with or without β clamp. Cy3-labelled 35-mer primer (X = T, G or 8-oxo-G) with 100-mer template DNA (N = A or C) having biotin/streptavidin at both ends (20 nM) was incubated with pol III* (10 nM) and β clamp (0, 10 or 100 nM) in the presence of 100 μM dNTPs. The reaction mixtures were incubated at room temperature for 1 min. The samples were analysed by denaturing polyacrylamide gel electrophoresis and visualized by the Molecular Imager. The alphabets shown in the figure represent: X, primer terminal base; N, template base; A, adenine; C, cytosine; G, guanine; T, thymine; oG, 8-oxo-G. The arrow indicates the position of primer. Although we have purified the primer DNA, there appears shorter primer DNAs, which were present below the position of the primer.
Article Snippet: Deoxyribonucleoside triphosphates (ultrapure grade) and 8-oxo-dGTP were purchased from Amersham Pharmacia Biotech (Buckinghamshire, UK) and TriLink BioTechnologies (San Diego, CA, USA) respectively.
Techniques: Incubation, Polyacrylamide Gel Electrophoresis, Purification