a
Exp#
|
b
Bkg (CMF × 10
−3
)
|
2 mM MgCl
2
|
0.4 mM MnCl
2
|
6 mM MnCl
2
|
0.25 mM CoCl
2
|
6 mM CoCl
2
|
0.4 mM ZnCl
2
|
|---|
c
1.6 mM free
|
0.15 mM free
|
5.6 mM free
|
0.07 mM free
|
5.6 mM free
|
0.15 mM free
|
|---|
(CMF × 10
−3
)
|
(CMF × 10
−3
)
|
(CMF × 10
−3
)
|
(CMF × 10
−3
)
|
(CMF × 10
−3
)
|
(CMF × 10
−3
)
|
|---|
d1 | 4/1893 | 11/1395 | 10/1297 | | | | 10/2993 |
2.1 | 7.9 (5.8) | 7.7 (5.6) | 3.3 (1.2) |
2 | 2/1246 | 8/1176 | 7/1067 | | 10/1054 | | 5/1301 |
1.6 | 6.8 (5.2) | 6.6 (5.0) | 9.5 (7.9) | 3.8 (2.2) |
3 | 3/3456 | 10/1755 | 10/2145 | | 16/1389 | | 14/3799 |
0.9 | 5.7 (4.8) | 4.7 (3.8) | 12 (11.1) | 3.7 (2.8) |
4 | 4/1250 | 10/1192 | 11/1257 | | 12/1007 | | |
3.2 | 8.4 (5.2) | 8.8 (5.6) | 12 (8.8) |
5 | 3/3113 | | | 160/1823 | 14/1324 | 101/1084 | |
0.96 | 88 (87) | 11 (10) | 93 (92) |
6 | 7/5866 | | | 249/1286 | | 90/1850 | |
1.2 | 194 (193) | 49 (48) |
7 | 3/2682 | | | 375/2740 | | 119/2503 | |
1.1 | 137 (136) | 48 (47) |
eAvg. ± S.D. | 1.6 ± 0.8 | 7.2 ± 1.7 | 7.4 ± 1.8 | 140 ± 53 | 11.1 ± 1.2 | 63 ± 26 | 3.6 ± 0.3 |
(5.3 ± 0.4) | (5.7 ± 1.6) | (139 ± 53) | (9.5 ± 1.4) | (62 ± 26) | (2.1 ± 0.8) |
fStatistics (see legend) | | 0.63 | 0.0034 | 0.0012 | 0.0059 | 0.00097 |
gRelative fidelity | | 1 | 0.93 | 0.038 | 0.56 | 0.085 | 2.5 |
-
aIndependent experiments performed at different times.
-
bIn background assays, the gapped plasmid was transformed into the bacteria allowing the bacterial polymerases to fill in the gap. Numbers shown are the “colony mutant frequency” (CMF) defined as white + faint blue colonies divided by total colonies. Refer to the Results and Methods sections for details.
-
cFree cation concentration under each condition was calculated as described in Materials and Methods using the dissociation constant for Mg2+ and ATP.
-
dNumbers shown on top are: (white + faint blue colonies)/total colonies. The bottom number is the colony mutant frequency (CMF) (see b above) for experiments under the listed condition. The CMF minus the background frequency from column two is in parentheses.
-
eAverages ± standard deviations from the experiments in the column are shown. Values in parentheses are after background subtraction.
-
fP-values shown were calculated using a standard Student’s t-test and the background subtracted values from each condition. All values were compared to the 2 mM MgCl2 condition. In order to address the effect of comparing multiple sample conditions on statistical significance, ANOVA analysis coupled with a Tukey’s honest significance of difference (Tukey HSD) test were conducted using the background subtracted values from samples and the calculator available online from Statistica:http://statistica.mooo.com/OneWay_Anova_with_TukeyHSD. The 6 mM MnCl2 and CoCl2 conditions were excluded from the analysis as their dramatically different magnitudes compared to other values complicates the analysis. Tukey analysis indicated highly significant differences (P < 0.01) for all conditions tested except 2 mM MgCl2 vs. 0.4 mM MnCl2, which was not significant, consistent with the insignificant P value (0.63) from the Student’s t-test.
-
gAll values are relative to the 2 mM Mg2+ average CMF-Bkg. value (0.0053). The 0.0053 value was divided by the average CMF-Bkg. value for each condition to determine relative fidelity. Higher numbers indicate greater fidelity.
