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

Table 5 Mismatched primer extension with Mg ²⁺ or Zn ²⁺

^a Cation	^b Base pair at the 3′ end	^c V _max	^d K _m	V _max / K _m	^e Standard extension efficiency, f _ext	^f Relative fidelity	^g P-value
^a Cation	^b Base pair at the 3′ end	%/min	μM	V _max / K _m	^e Standard extension efficiency, f _ext	^f Relative fidelity	^g P-value
^hMg²⁺	C.G	21.4 ± 2.0	0.37 ± 0.13	57.8 ± 19.2	1
	G.C	39.9 ± 14.5	0.27 ± 0.13	147.8 ± 40.6	1
	C.T	22.5 ± 2.1	396 ± 28	5.7 (±0.9) x 10⁻²	9.9 (±1.7) x 10⁻⁴
	C.A	11.1 ± 2.9	445 ± 188	2.5 (±0. 57) x 10⁻²	4.3 (±0.94) x 10⁻⁴
	C.C	0.70 ± 0.29	77 ± 14	9.1 (±2.2) x 10⁻³	1.6 (±0.38) x 10⁻⁴
	G.T	18.6 ± 3.3	157 ± 44	0.12 ± 0.03	8.1 (±2.5) x10⁻⁴
	G.A	0.95 ± 0.04	196 ± 50	4.8 (±1.6) x 10⁻³	3.2 (±1.0) x 10⁻⁵
Zn²⁺	C.G	0.62 ± 0.23	0.04 ± 0.01	15.5 ± 5.8	1	1
	G.C	0.39 ± 0.10	0.04 ± 0.01	9.8 ± 1.9	1	1
	C.T	2.9 ± 1.0	591 ± 63	4.9 (±2. 3) x 10⁻³	3.2 (±1.6) x 10⁻⁴	3.1	0.026
	C.A	3.3 ± 1.0	1240 ± 186	2.7 (±1.3) x 10⁻³	1.7 (±0.75) x 10⁻⁴	2.5	0.029
	C.C	0.50 ± 0.14	914 ± 457	5.4 (±4.6) x 10⁻⁴	3.5 (±3.4) x 10⁻⁵	4.6	0.023
	G.T	6.8 ± 1.8	1223 ± 568	5.6 (±2.8) x 10⁻³	5.7 (±3.2) x 10⁻⁴	1.4	0.375
	G.A	N.D.

^aThe extension reactions were carried out in either 2 mM fee 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 mismatch extension sequences in Figure 2. In this assay primers with a matched C.G or a mismatched C.T, C.A, or C.C at the 3′ end were extended on one seqeunce. A second sequence with a matched G.C or mismatched G.T or G.A was also used.
^c V _max is the maximum velocity of extending each primer- template hybrid. See Materials and Methods for a description.
^dRefers to the K _m of the next correct nucleotide being added (i.e. dCTP for C.G, C.T, C.A, and C.C or dGTP for G.C, G.T, and G.A extensions).
^e f _ext is the ratio of {V _max/K _m (mismatch)}/{V _max/K _m (match)}.
^fFidelity values for misextension in Zn²⁺ are relative to the same mismatch using Mg²⁺. Determinations were made by dividing the standard extension efficiency in Mg²⁺ by the same parameter in Zn²⁺. Higher values indicate greater fidelity.
^gValues were calculated using a standard Student’s t-test. Standard extension efficiency values from experiments in Zn²⁺ were compared against the Mg²⁺condition for the same mismatch.
^hValues for Mg²⁺ were taken from (33).

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