Our earlier study highlighted the tendency of full-length human Pol ν to generate G to A substitutions via misincorporation of dTTP opposite template guanine (25
). We began this study by confirming this error signature for a slightly abbreviated derivative of Pol ν (the eventual goal being structural studies), designated Pol ν-77 (36
). We used the M13mp2 forward mutation assay, which detects a wide range of sequence changes in the lacZ
gene in a variety of sequence contexts (35
). The DNA products of gap-filling synthesis at pH 8.8 yielded an average lacZ
mutant frequency of 15% (), comparable to the 18% value reported previously for the full-length protein (25
). In addition to similar error rates, the truncated and full-length Pol ν proteins have similar processivity (compare in reference (25
) and in (36
)). We also examined the fidelity at pH 7.5, the pH used in our kinetic studies below. Pol ν is more accurate at pH 7.5, as revealed by a 4-fold lower average mutant frequency of 3.5% (). Higher accuracy at lower pH is also a characteristic of other Family A DNA polymerases, including Taq
) and the large Klenow fragment of E. coli
pol I (42
). When 204 independent lacZ
mutants were sequenced from reactions performed at pH 7.5, a variety of single base changes were observed (). These were distributed throughout the lacZ
target sequence (), in a pattern similar to that seen earlier with full-length human Pol ν at pH 8.8 (25
). Substitutions predominated, and the vast majority were G to A changes, yielding an average error rate of 22 × 10−4
for stable misincorporation of dTTP opposite the 22 template guanines where this event results in a change in plaque color (35
). The second most common error was the mismatch involving the same two bases but differing with respect to the templating versus incoming base, i.e., the T-dGTP mismatch (average error rate of 2.0 × 10−4
, ). We have previously compared Pol ν specific error rates to the error rates of other DNA polymerases (see in (25
Summary of sequence changes generated by Pol ν-77
Figure 2 Kinetics of Pol ν-77. Panel A shows the kinetics of incorporation of dATP into the 13/19mer-T substrate (). The reactions contained 5 nM DNA primer termini, 100 μM dATP and the indicated concentrations of Pol ν-77 (measured (more ...)
Figure 4 Regions of the A-family DNA polymerase structure where Pol ν shows interesting differences from the classical A-family conserved motifs. Panel A shows the structure of the polymerase domain of the ternary (Pol-DNA-dNTP) complex of Klentaq (3KTQ, (more ...)
Figure 1 Spectrum of errors generated by human Pol ν-77 at pH 7.5. The 407 template nucleotides within the single-strand gap of the M13mp2 substrate are shown as 5 lines of the template sequence, bracketed by 5 base pairs at either end to indicate the (more ...)
As before (25
), G to A mutation rates varied by sequence context. The highest mutation rate for a phenotypically detectable site was observed at template guanine number +165 (designated the G(165) hotspot), where 38 substitutions were observed among 204 sequenced lacZ
mutants. This corresponds to an error rate of 1.1%; because of this high error rate, the G(165) hotspot sequence was chosen for our kinetic analysis. Additionally, we measured dNTP insertion kinetics at the neighboring guanine, G(164), which is also a hotspot. Although substitutions at G(164) are phenotypically silent, two G to A substitutions were observed as silent hitchhikers among 204 sequenced lacZ
mutants, corresponding to an error rate of 1.6%, similar to the error rate at G(165) 2
. For comparison, we analyzed Pol ν kinetics at a mutational coldspot, G(−66), selected because only three G to A changes were detected at this phenotypically-detectable position, corresponding to an error rate of 0.09%. dNTP misinsertion.
The design of the kinetics experiments was influenced by the properties of the Pol ν-77 preparations. Purification of Pol ν-77 is challenging and the purified protein (36
) was typically not fully active, as shown by active-site titration using the measurement of burst amplitudes in primer extension reactions (). An alternative method using DNA binding to assess the concentration of active Pol ν-77 is illustrated in Supplementary Figure S1
. Measurements by the two methods were in good agreement. The Pol ν-77 preparation used in the majority of the kinetics experiments contained 23% active polymerase. As described in Methods, we measured reaction rates under burst conditions, with the concentration of active Pol ν-77 about 3-fold less than the concentration of the DNA substrate. This had the advantage of conserving our stocks of enzyme and also avoided the problem that high concentrations of some Pol ν-77 preparations inhibited the reaction.
We measured misinsertion by Pol ν-77 on several different DNA substrates (). The 13/19-mer duplex is a DNA substrate that we have used routinely in recent kinetic studies of Klenow fragment (38
). We compared misinsertions at a template T and a template G in this sequence. The results with Klenow fragment (, line 2 of each section) were in good agreement with data previously obtained on a different DNA sequence (reproduced in , line 1 of each section) (44
). As described above, three other DNA substrates were derived from the lacZ
target sequence, in order to study dTTP misinsertion opposite template guanines in three different sequence contexts: the G(164) and G(165) hotspots and the G(−66) coldspot. In each case, we measured the rates of correct and mismatched dNTP incorporation at a series of dNTP concentrations and calculated kpol
. shows an example of the data obtained for T misinsertion opposite G(164). The misinsertion data obtained for Pol ν-77, with selected Klenow fragment misinsertions for comparison, are shown in . The final column of compares the efficiencies of correct and incorrect dNTP addition on each substrate, giving the selectivity in favor of the correct complementary dNTP.
Kinetics of dNTP insertion catalyzed by Pol ν-77 and by exonuclease-deficient Klenow fragment
The misinsertion data for the 13/19-mer substrate fit well with the overall mutational data for both Klenow fragment and Pol ν. For example, Klenow fragment has 3-fold lower discrimination against T-dGTP errors (1.3 × 10−4
, bottom of ) compared to G-dTTP errors (3.4 × 10−4
, top of ), consistent with its higher G-dTTP error rate in the M13mp2 forward mutation assay (10
). For both T-dGTP and G-dTTP errors, the individual kinetic constants for Klenow fragment show that the selectivity in favor of the correct base pair is made up of ~ 10-fold discrimination in dNTP binding and ~ 103
-fold discrimination in the rate of misincorporation, with the kinetic differences between correctly paired and mispaired substrates slightly larger in the case of G-dTTP errors. For Pol ν, the most frequent error in the M13mp2 assay is G-dTTP, whose rate is 11-fold higher than that of T-dGTP (). Again, this is reflected in the kinetic data, where we observe a 13-fold difference in discrimination against the two mismatches (selectivity values of 38 for G-dTTP versus 510 for T-dGTP, third lines in top and bottom sections of ).
What accounts for the low fidelity of Pol ν for G-dTTP errors? Both Klenow fragment and Pol ν have similar kinetic constants for misinsertion of dTTP opposite template G (, top right). However, correct dCTP insertion by Pol ν opposite template G is ~ 103-fold less efficient than is correct dCTP insertion catalyzed by Klenow fragment (, top left). The difference in the efficiency of correct incorporation therefore translates directly to ~ 103-fold lower selectivity of Pol ν against G-dTTP mismatches, compared with Klenow fragment. By contrast, at template T, dGTP misinsertion by Pol ν is ~ 260-fold less efficient than that of Klenow fragment (, lower right, compare 950 vs. 3.7). This difference moderates the effect of the large (6,300-fold) difference in the efficiency of correct (T-dATP) incorporation (, lower left, compare 1.2 × 107 to 1.9 × 103), resulting in ~ 24-fold lower selectivity of Pol ν against T-dGTP errors.
The kinetic data for G-dTTP misinsertion by Pol ν on the lacZ sequences also parallel the mutational data. Error rates in the M13mp2 fidelity assay are high at template G(165) and G(164) and lower at template G(−66). Likewise, selectivity against dTTP misinsertion () is at least 20-fold higher opposite template G(−66) (selectivity factor of 520) than opposite templates G(165) and G(164) (selectivity factors of 22 and 25, respectively). The different selectivity values derive from some very interesting sequence-dependent differences in individual kinetic constants. First, the binding constants for both correct dCTP and incorrect dTTP are 15- to 20-fold weaker at the G(−66) position as compared to the G(164) and G(165) positions. At G(−66), where the error rate is lowest, incorporation of correct dCTP is ≈ 16-fold faster than misinsertion of dTTP (, kpol of 0.28 and 0.018, respectively). In contrast, at the G(164) and G(165) hotspots, the kpol values for correct dCTP incorporation are slower than at G(−66), and similar to those for misinsertion of dTTP at these same hotspot sequences. Therefore, the lower fidelity of Pol ν for G-dTTP mismatches in one sequence context as compared to another can be attributed largely to differences in the kinetics of correct G-dCTP incorporation, particularly the rate of dCTP addition, since the ratio of dCTP and dTTP binding constants is very similar at all three sequences. The kinetic constants for the template G in the 13/19-mer DNA substrate resemble to some extent those of the G(−66) sequence, with weak binding constants and a fast rate of G-dCTP incorporation.