Human Pol ε-dependent replication errors and the influence of mismatch repair on their correction

Abstract

Mutations in human DNA polymerase (Pol) ε, one of three eukaryotic Pols required for DNA replication, have recently been found associated with an ultramutator phenotype in tumors from somatic colorectal and endometrial cancers and in a familial colorectal cancer. Possibly, Pol ε mutations reduce the accuracy of DNA synthesis, thereby increasing the mutational burden and contributing to tumor development. To test this possibility in vivo, we characterized an active site mutant allele of human Pol ε that exhibits a strong mutator phenotype in vitro when the proofreading exonuclease activity of the enzyme is inactive. This mutant has a strong bias toward mispairs opposite template pyrimidine bases, particularly T • dTTP mispairs. Expression of mutant Pol ε in human cells lacking functional mismatch repair caused an increase in mutation rate primarily due to T • dTTP mispairs. Functional mismatch repair eliminated the increased mutagenesis. The results indicate that the mutant Pol ε causes replication errors in vivo, and is at least partially dominant over the endogenous, wild type Pol ε. Since tumors from familial and somatic colorectal patients arise with Pol ε mutations in a single allele, are microsatellite stable and have a large increase in base pair substitutions, our data are consistent with a Pol ε mutation requiring additional factors to promote tumor development.

Keywords: DNA polymerase; DNA replication; Mismatch repair; Mutagenesis.

Figure 1. Effects on of the M630G active site mutation in human Pol ε on DNA synthesis and 3′-5′ exonuclease activities

(A) Schematic of the 1189 aa human Pol ε construct used for in vitro experiments in this study and previously [35, 43]. The six conserved exonuclease (Exo) and polymerase (Pol) motifs are shown. The position of the M630G active site mutation is indicated. (B) 500 ng of purified M630G-Pol ε^exo+ and M630G-Pol ε^exo− enzymes were run on 10% denaturing PAGE gels and stained by Coomassie. Molecular weight marker (MW) is shown with selected molecular weights indicated in kDa. (C) Primer extension assays of M630G-Pol ε. The relative DNA synthesis activities of wild type (M630) and mutant (M630G) human Pol ε enzymes were compared in both the exonuclease-proficient (exo⁺) and – deficient (exo⁻) backgrounds. Enzyme (1 nM) was incubated with all four dNTPs (25 μM each), 8 mM Mg²⁺ and a duplex DNA substrate containing a 19-mer deoxyribonucleotide primer hybridized to a complementary 45-mer template (100 nM). Reactions were carried out as described in the Materials and Methods. Reactions were performed at 37°C and started by the addition of enzyme. Aliquots were removed at 2, 5 and 10 minutes and products were resolved on a 12% denaturing acrylamide gel. Control substrate with no enzyme added is shown (−). (D) 3′-5′ exonuclease assays of M630G-Pol ε. The relative 3′-5′ exonuclease activities of wild type (M630) and mutant (M630G) human Pol ε were also compared in both the exonuclease-proficient (exo⁺) and – deficient (exo⁻) backgrounds. Enzyme was incubated with 8 mM Mg²⁺ and a ssDNA 18-mer deoxyribonucleotide substrate (100 nM) in the absence of dNTPs. Reactions were carried out as described in the Materials and Methods. Reactions were performed at 37°C and started with the addition of 0.5 nM enzyme. Aliquots were removed at 2, 5 and 10 minutes. Control substrate with no enzyme added is shown (−). Products were resolved on a 12% denaturing acrylamide gel.

Figure 1. Effects on of the M630G active site mutation in human Pol ε on DNA synthesis and 3′-5′ exonuclease activities

(A) Schematic of the 1189 aa human Pol ε construct used for in vitro experiments in this study and previously [35, 43]. The six conserved exonuclease (Exo) and polymerase (Pol) motifs are shown. The position of the M630G active site mutation is indicated. (B) 500 ng of purified M630G-Pol ε^exo+ and M630G-Pol ε^exo− enzymes were run on 10% denaturing PAGE gels and stained by Coomassie. Molecular weight marker (MW) is shown with selected molecular weights indicated in kDa. (C) Primer extension assays of M630G-Pol ε. The relative DNA synthesis activities of wild type (M630) and mutant (M630G) human Pol ε enzymes were compared in both the exonuclease-proficient (exo⁺) and – deficient (exo⁻) backgrounds. Enzyme (1 nM) was incubated with all four dNTPs (25 μM each), 8 mM Mg²⁺ and a duplex DNA substrate containing a 19-mer deoxyribonucleotide primer hybridized to a complementary 45-mer template (100 nM). Reactions were carried out as described in the Materials and Methods. Reactions were performed at 37°C and started by the addition of enzyme. Aliquots were removed at 2, 5 and 10 minutes and products were resolved on a 12% denaturing acrylamide gel. Control substrate with no enzyme added is shown (−). (D) 3′-5′ exonuclease assays of M630G-Pol ε. The relative 3′-5′ exonuclease activities of wild type (M630) and mutant (M630G) human Pol ε were also compared in both the exonuclease-proficient (exo⁺) and – deficient (exo⁻) backgrounds. Enzyme was incubated with 8 mM Mg²⁺ and a ssDNA 18-mer deoxyribonucleotide substrate (100 nM) in the absence of dNTPs. Reactions were carried out as described in the Materials and Methods. Reactions were performed at 37°C and started with the addition of 0.5 nM enzyme. Aliquots were removed at 2, 5 and 10 minutes. Control substrate with no enzyme added is shown (−). Products were resolved on a 12% denaturing acrylamide gel.

Figure 2. Base substitution and frameshift error rates for human M630G-Pol ε^exo−

(A) Error rates for base pair substitutions (BPS), overall frameshifts (FS), −1 and +1 frameshifts. Error rates were calculated as described [39]. Error rates are shown for the exonuclease-deficient (light gray bars) and – proficient (gray bars) M630G active site mutant Pol ε. Included for comparison are error rates for the exonuclease-deficient M630 Pol ε that we characterized previously (black bars, [35]). (B) Fidelity of individual base pair substitutions for M630G-Pol ε^exo− and M630-Pol ε^exo−. Error rates for each of the 12 possible mispairs for the exonuclease-deficient forms of M630G-Pol ε (gray bars, this study) and M630-Pol ε (black bars, [35]) were calculated as described [39]. (C) Individual in vitro replication errors made by the M630G-Pol ε^exo− enzyme in the forward mutation assay are indicated above the reference lacZ sequence. Single base deletions are shown as empty triangles and single base insertions are shown as filled triangles.

Figure 2. Base substitution and frameshift error rates for human M630G-Pol ε^exo−

(A) Error rates for base pair substitutions (BPS), overall frameshifts (FS), −1 and +1 frameshifts. Error rates were calculated as described [39]. Error rates are shown for the exonuclease-deficient (light gray bars) and – proficient (gray bars) M630G active site mutant Pol ε. Included for comparison are error rates for the exonuclease-deficient M630 Pol ε that we characterized previously (black bars, [35]). (B) Fidelity of individual base pair substitutions for M630G-Pol ε^exo− and M630-Pol ε^exo−. Error rates for each of the 12 possible mispairs for the exonuclease-deficient forms of M630G-Pol ε (gray bars, this study) and M630-Pol ε (black bars, [35]) were calculated as described [39]. (C) Individual in vitro replication errors made by the M630G-Pol ε^exo− enzyme in the forward mutation assay are indicated above the reference lacZ sequence. Single base deletions are shown as empty triangles and single base insertions are shown as filled triangles.

Figure 3. Effect of M630G-Pol ε^exo− expression in human cells on mutant frequency

(A) HCT-116 mismatch repair-deficient cells were grown to 70% confluence in 100 mm dishes and then transfected with 3 μg of the indicated vector DNA. After 48 hours, cells were harvested and lysed in buffer containing 1% Triton X-100. Cell extracts were probed by Western blot (WB) using antibodies against FLAG to detect the recombinant Pol ε, as well as against GFP and actin (left). Duplicate cell lysate samples were probed by WB using antibodies against the catalytic subunit of Pol ε (p261) to detect the total combined recombinant and endogenous Pol ε (right). Pol ε p261 levels were quantitated (NIH ImageJ) and are shown relative to levels in untransfected cells. (B) To measure mutant frequencies, cells were transfected as described in (A) with the indicated constructs, then grown for 48 hours and trypsinized. 500 cells were seeded in duplicate into media lacking 6-thioguanine (6-TG) and grown for 5–7 days to determine plating efficiency. 4.5 × 10⁵ cells were seeded in triplicate into media containing 6-TG to select for HPRT1⁻ mutant cells and grown for 12–14 days. Colonies were then stained with crystal violet and counted. Mutant frequency was calculated by the following equation: (# 6-TG resistant colonies)/[(# 6-TG seeded cells) × (plating efficiency # scored colonies)/(plating efficiency # cells seeded)]. Colonies were defined as ≥ 50 cells. Average values and standard deviations from 3 independent experiments were calculated and plotted relative to HCT-116 cells transfected with empty vector. P-values are shown above each comparison where the difference was found to be significant (*, p<0.05; **, p<0.005).

Figure 4. Generation of cell lines stably expressing M630G-Pol ε^exo−

(A) Clones were evaluated for expression of M630G-Pol ε^exo− by Western blot. Cells were harvested and lysed in buffer containing 1% Triton X-100. Cell extracts were probed by Western blot using antibodies against FLAG, Pol ε catalytic subunit (α-p261), actin and GFP.