Table 1.

Steady-state kinetics of single nucleotide insertion opposite dG, 2′-F dG, and N⁷-CH₃ 2′-F dG by hpol η

The oligonucleotides used were as follows,

5′-FAM-CGGGCTCGTAAGCGTCAT-3′ 3′-GCCCGAGCATTCGCAGTAXTACT-5′

where X represents dG, 2′-F dG, or N⁷-CH₃ 2′-F dG.

Template base	dNTP	kcat	Km	kcat/Km	fa
		min−1	μm	μm−1 min−1
dG	dCTP	2.65 ± 0.11	0.45 ± 0.13	5.9 ± 1.7	1
2′-F dG	dCTP	1.77 ± 0.05	0.23 ± 0.06	7.7 ± 2.0	1
N7-CH3 2′-F dG	dCTP	1.65 ± 0.04	0.43 ± 0.07	3.8 ± 0.6	1
dG	dATP	0.76 ± 0.06	12 ± 5	0.06 ± 0.03	0.01
2′-F dG	dATP	1.1 ± 0.1	19 ± 6	0.06 ± 0.03	0.01
N7-CH3 2′-F dG	dATP	0.97 ± 0.04	14 ± 2	0.07 ± 0.01	0.018
dG	dGTP	0.65 ± 0.05	4.0 ± 1.7	0.16 ± 0.07	0.027
2′-F dG	dGTP	0.84 ± 0.06	2.7 ± 1.2	0.31 ± 0.14	0.040
N7-CH3 2′-F dG	dGTP	0.40 ± 0.06	7.5 ± 6.3	0.05 ± 0.04	0.013
dG	dTTP	1.42 ± 0.14	32 ± 11	0.04 ± 0.01	0.0068
2′-F dG	dTTP	0.96 ± 0.10	38 ± 15	0.03 ± 0.01	0.0033
N7-CH3 2′-F dG	dTTP	0.62 ± 0.05	4.6 ± 2.2	0.14 ± 0.07	0.037

^a Misincorporation frequency f = (k_cat/K_m)_incorrect/(k_cat/K_m)_correct.