Low-fidelity DNA synthesis by human DNA polymerase theta

Abstract

Human DNA polymerase theta (pol or POLQ) is a proofreading-deficient family A enzyme implicated in translesion synthesis (TLS) and perhaps in somatic hypermutation (SHM) of immunoglobulin genes. These proposed functions and kinetic studies imply that pol may synthesize DNA with low fidelity. Here, we show that when copying undamaged DNA, pol generates single base errors at rates 10- to more than 100-fold higher than for other family A members. Pol adds single nucleotides to homopolymeric runs at particularly high rates, exceeding 1% in certain sequence contexts, and generates single base substitutions at an average rate of 2.4 x 10(-3), comparable to inaccurate family Y human pol kappa (5.8 x 10(-3)) also implicated in TLS. Like pol kappa, pol is processive, implying that it may be tightly regulated to avoid deleterious mutagenesis. Pol also generates certain base substitutions at high rates within sequence contexts similar to those inferred to be copied by pol during SHM of immunoglobulin genes in mice. Thus, pol is an exception among family A polymerases, and its low fidelity is consistent with its proposed roles in TLS and SHM.

Figure 1.

Spectrum of errors generated by human pol θ. The 407 template nucleotides within the single-strand gap of the M13mp2 substrate are shown as five lines of the template sequence. Letters above the target sequence indicate base substitutions. Deletion of a base is depicted by an open triangle, whereas addition of a base is represented by a closed inverted triangle above the target sequence. ‘Red’ characters represent phenotypically detectable changes in the gap region while ‘black’ characters represent phenotypically undetectable changes found in association with detectable changes. ‘Gray’ arrows indicate hot spots of template A or T base substitutions (−36, +19, +113 and +131) and base substitution hot spots (+131 and +136) +1 represents the first transcribed nucleotide of the lacZα-complementation region.

Figure 2.

Pol θ indel error rates as a function of homopolymeric run length. Error rates are the number of all observed single-base deletions and single base-additions (Table 1 and Figure 1) divided by the total number of template nucleotides present in runs of the indicated length indicated in the figure among 200 sequenced lacZ clones generated by pol θ.

Figure 3.

Processive synthesis by Klenow exo⁻ and pol θ. Reactions were performed as described in Materials and Methods section. (A) Representative phosphor image of the reaction products of processive DNA synthesis resolved on a 12% denaturing polyacrylamide gel. Lanes 2–5 represent primer extension products by Klenow exo⁻ at 1, 3, 4 and 5 min. Lanes 7–11, primer extension products by pol θ at 2, 5, 10, 15 and 30 min. Lanes 1 and 6 represent negative control reactions. The numbers on the right indicate the positions along the lacZ gene. (B) Termination probability per nucleotide at each template position, 144 to 127. Numbers along the Y-axis indicate nucleotide positions along the lacZ gene. The termination probability is defined as the amount of product of a given length divided by the sum of products of that length plus all greater length products. The values plotted are the average of three values for pol θ (for time points 5, 10, and 15 min for pol θ:DNA at one ratio) and an average of three values for Klenow exo⁻ (time points 3, 4 and 5 min at one ratio) and the error bars represent the standard deviation. Error rates for certain sequence contexts (CCCCC at positions 132–136 and TTT at positions 137–139) are shown for both pol θ and Klenow exo^– (21).

Figure 4.

Pol θ error rates compared to other DNA polymerases. (A) Single-base deletions (B) Single-base additions (C) Base substitutions. Family Y: hPol κ and hPol η error rates are taken from (20). Family X: hPol β and hPol λ error rates are taken from (57). Family B: hPol α error rates are unpublished results from Kokoska, R.J. and Kunkel, T.A., yPol δ (Exo–) error rates are taken from (58), yPol ɛ (Exo–) error rates are taken from (59) and yPol ζ error rates are taken from (50). Family A: hPol θ and hPol ν data are from this study. For hPol ν, at neutral pH, a mutation frequency of 2.6% was obtained (64 mutant plaques from a total of 6432). Of these, 22 clones were sequenced and as with the previous study (24), these clones show a preference for G to A base substitutions. hPol γ (Exo–) (+p140 and p55) error rates are from (23) and Kf (Exo−) error rates are taken from (21).