Alternative divalent cations (Zn2+, Co2+, and Mn2+) are not mutagenic at conditions optimal for HIV-1 reverse transcriptase activity

Table 4 Running-start misincorporation assay of various mismatches with Mg ²⁺ or Zn ²⁺

^a Cation	^b Base pair	^c V _max,rel	^d K _m	V _max/K _m	^e Misinsertion ratio, f _ins	^f Relative fidelity	^g P-value
^a Cation	^b Base pair	^c V _max,rel	μM	μM ⁻¹	^e Misinsertion ratio, f _ins	^f Relative fidelity	^g P-value
^hMg²⁺	C.G	2.9 ± 1.1	0.68 ± 0.33	4.3 ± 1.3	1
	G.C	1.4 ± 0.26	1.2 ± 0.64	1.2 ± 0.6	1
	C.T	0.55 ± 0.14	1863 ± 670	2.9 (±0.33) x 10⁻⁴	6.7 (±2.6) x 10⁻⁵
	C.A	0.50 ± 0.06	825 ± 217	6.1 (±1.1) x 10⁻⁴	1.4 (±0.3) x 10⁻⁴
	C.C	0.09 ± 0.07	312 ± 166	2.9 (±0.8) x 10⁻⁴	6.7 (±1.9) x 10⁻⁵
	G.T	0.36 ± 0.16	242 ± 50	1.5 (±0.9) x10⁻³	1.3 (±0.8) x10⁻³
	G.A	0.38 ± 0.11	1515 ± 85	2.5 (±0.6) x 10⁻⁴	2.1 (±0.5) x 10⁻⁴
Zn²⁺	C.G	8.2 ± 5.5	0.19 ± 0.09	43 ± 11	1
	G.C	5.8 ± 5.1	5.1 ± 4.7	1.3 ± 0.6	1
	C.T	0.19 ± 0.07	244 ± 102	7.8 (±0.97) x 10⁻⁴	1.8 (±0.8) x 10⁻⁵	3.7	0.028
	C.A	0.55 ± 0.33	518 ± 43	1.1 (±0.6) x 10⁻³	2.5 (±1.6) x 10⁻⁵	5.6	0.0023
	C.C	0.05 ± 0.01	224 ± 104	2.2 (±1.4) x 10⁻⁴	5.1 (±5.6) x 10⁻⁶	13.1	0.029
	G.T	0.2 ± 0.08	914 ± 378	2.2 (±0.7) x 10⁻⁴	1.6 (±0.6) x 10⁻⁴	8.1	0.038
	G.A	N.D.

^aThe extension reactions were carried as described in Materials and Methods using either 2 mM free Mg²⁺ or 0.4 mM free Zn²⁺. Free cation concentration was calculated as described in Materials and Methods using the dissociation constant for Mg²⁺ and ATP. All values are averages from at least 3 experiments ± standard deviation.
^bRefer to the running-start sequences in Figure 2. The particular mismatch that was measured after incorporation of a run of A’s over a run of T’s on the template is shown in the column
^c V _max,rel = I _i/I _i − 1 where I _i is the sum of band intensities at the target site and beyond, I _i − 1 is the intensity of the band prior to the target band. See Materials and Methods for a description.
^dRefers to the K_m of the nucleotide being incorporated at the target site (e.g. dGTP for C.G and dATP for C.A).
^e f _ins is the ratio of {V _max/K _m (mismatch)}/{V _max/K _m (match)}.
^fFidelity values for misincorporation in Zn²⁺ are relative to the same mismatch using Mg²⁺. Determinations were made by dividing the misinsertion ratio in Mg²⁺ by the ratio in Zn²⁺. Higher values indicate greater fidelity.
^gValues were calculated using a standard Student’s t-test. Misinsertion ratio values from experiments in Zn²⁺ were compared against the Mg²⁺ condition for the same misincorporation.
^hValues for Mg²⁺ were taken from (33).

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