Figure 2.

Faithful DNA proofreading and polymerase activities of engineered B35-HhH DNA polymerase. Primer extension assays with an oligonucleotide template/primer duplex substrate (1 nM) as depicted above. In the scheme, the template is represented in grey, primer in blue and incorporated nucleotides during the primer extension in black. Reactions were performed in the presence 10 nM of the indicated DNA polymerase and increasing concentration of dNTPs—25, 50, 100 and 500 nM (A), or in a time-course experiment during 15, 30, 60 or 120 s with 100 nM dNTPs (B). Nucleotide insertion fidelity was analysed on running extension assays triggered with 10 mM MgCl₂ (C). 15-, 17- and 19-mer or 15 and 33-mer oligonucleotides (OL15, OL17, OL19 and OL33, Table S1) were loaded as size markers (lane M). Relative misinsertion rate at each dATP concentration (D) was determined from three independent experiments. See “Materials and methods” for details.