Table 1.

Pre-steady state kinetic parameters for deoxy- vs ribonucleotide incorporation.

Template	PrimPol	NTP or dNTP	Km (µM)	kcat (s−1)	Catalytic efficiency kcat/Km (s−1 µM−1)	Discriminationa (NTP vs dNTP)	Improvedb NTP preference	Improvedc NTP Binding
dG	WT	CTP	9.08 ± 0.87	(3.08 ± 0.04) · 10−3	(0.34 ± 0.04) · 10−3	0.05	32-fold	13-fold
		dCTP	0.84 ± 0.05	(5.83 ± 0,11) · 10−3	(6.94 ± 0.57) · 10−3
	Y100H	CTP	0.69 ± 0.08	(3.24 ± 0.11) · 10−3	(4.73 ± 0.73) · 10−3	1.58
		dCTP	0.92 ± 0.20	(2.75 ± 0.17) · 10−3	(3.00 ± 0.83) · 10−3
dT	WT	ATP	38.68 ± 3.64	(0.97 ± 0.02) · 10−3	(0.03 ± 0.003) · 10−3	0.01	72-fold	36-fold
		dATP	0.78 ± 0.12	(3.18 ± 0.13) · 10−3	(4.05 ± 0.80) · 10−3
	Y100H	ATP	1.06 ± 0.12	(2.67 ± 0.09) · 10−3	(2.51 ± 0.36) · 10−3	0.72
		dATP	0.65 ± 0.21	(2.26 ± 0.18) · 10−3	(3.47 ± 1.42) · 10−3
8oxodG	WT	CTP	34.36 ± 2.70	(1.72 ± 0.04) · 10−3	(0.05 ± 0.005) · 10−3	0.01	173-fold	29-fold
		dCTP	0.83 ± 0.13	(3.91 ± 0.19) · 10−3	(4.72 ± 0.98) · 10−3
	Y100H	CTP	1.17 ± 0.10	(2.36 ± 0.07) · 10−3	(2.01 ± 0.23) · 10−3	1.73
		dCTP	2.56 ± 0.65	(2.98 ± 0.35) · 10−3	(1.16 ± 0.43) · 10−3
	WT	ATP	20.86 ± 5.68	(0.62 ± 0.03) · 10−3	(0.03 ± 0.01) · 10−3	0.01	75-fold	21-fold
		dATP	0.75 ± 0.09	(3.72 ± 0.12) · 10−3	(4.95 ± 0.73) · 10−3
	Y100H	ATP	1.01 ± 0.22	(2.49 ± 0.17) · 10−3	(2.47 ± 0.72) · 10−3	0.75
		dATP	0.75 ± 0.19	(2.45 ± 0.17) · 10−3	(3.27 ± 1.06) · 10−3

WT PrimPol or Y100H mutant kinetic parameters (Km, kcat, and catalytic efficiency) were calculated for the insertion of single nucleotides (CTP, dCTP, ATP and dATP) opposite a templating G, T or 8oxoG, as described in Methods. ^aNTP/dNTP discrimination ratio is calculated by dividing the catalytic efficiency of ribo vs deoxy versions of a given nucleotide. ^bImproved NTP preference ratio (Y100H mutant relative to WT PrimPol) is calculated by dividing the discrimination values for a given nucleotide. ^cImproved NTP binding ratio (Y100H mutant relative to WT PrimPol) is calculated by dividing the Km values for a given ribonucleotide.