Screening pol α mutants.
DNA polymerases from many different species have a structurally conserved sequence in the palm region (2
) (Fig. ). In this study, S. cerevisiae
DNA pol α mutants with altered amino acids were isolated using a genetic complementation assay and the temperature-sensitive strain pol1-17
. Active pol α mutants were identified with single or multiple amino acid substitutions (Fig. ). These mutants were rescreened by growing cells in the absence of tryptophan such that cell growth required reversion or suppression of a trp1
amber codon. The mutation rates for most of the mutants fell into a range of values 0.6- to 2.5-fold higher than those for the WT (data not shown). The experiments reported here were used to examine the properties of pol α L868F, whose spontaneous mutation rate for the pol1-17
strain was 8.7-fold higher than that of WT pol α.
FIG. 2. DNA polymerase motif A. (A) Conserved motifs A, B, and C are illustrated schematically. (B) Amino acid sequences of family A, family B, and family Y DNA polymerases are compared. Conserved amino acids are boxed. Phe34 in S. cerevisiae pol η is (more ...) Fidelity of S. cerevisiae L868F pol α.
In experiments designed to discover whether inaccurate DNA synthesis caused the mutator phenotype, WT pol α and Leu868 mutants carrying Phe, Met, Trp, or Val substitutions were purified and characterized. The specific activity of these mutants was similar to that of WT pol α (Table ), but their in vitro mutation rate was significantly higher than that of the WT. In a lacZα forward mutation assay, WT pol α had a mutation frequency of 3.4 × 10−3 and L868F pol α had a 138-fold-higher mutation frequency (470 × 10−3) (Table ).
Error frequencies for WT and mutant pol α in a lacZα forward mutation assay
The mutation spectra of L868F mutant and WT pol α confirmed the observed difference in mutant frequencies. In the mutant phages, WT pol α made approximately one mutation in the target LacZα sequence; in contrast, L868F pol α made an average of nine errors in the same target sequence. Calculating on the basis of 1,234 mutations, the spontaneous error rate of L868F pol α was estimated to be 3 per 100 nucleotides, 570-fold higher than the results seen with the WT (Table ). The spontaneous errors exhibited the following characteristics. (i) Base substitution errors made by L868F pol α were randomly distributed throughout the target DNA sequence and not clustered in “hot spots” as were those made by WT pol α (Fig. ). (ii) In vitro, L868F pol α generated 43% transversion mutations and 44% transition mutations whereas WT pol α generated 67% transitions and 13% transversions (Table ). (iii) The relative frequencies of L868F pol α base substitution errors were C→T (21%) > A→T (12%) > G→T (7.9%), and the frequency of N→T substitutions was 41%. Thus, the rate of dAMP misincorporation was considerably higher than that of misincorporation of other nucleotides (Table ). (iv) L868F pol α also generated frameshift errors, with a 930-fold-higher rate of insertions and a 320-fold-higher rate of deletions than WT pol α (Table ). (v) WT pol α preferentially made single-base deletions at run sequences (18/18), while more than half (36/64) of the single-base deletions made by L868F pol α were in nonrun sequence. (vi) L868F pol α generated tandem base deletions as well as large deletions of 30 to 50 nucleotides.
Types of errors by S. cerevisiae WT and L868F pol α in a lacZα forward mutation assay
FIG. 3. S. cerevisiae L868F DNA pol α mutation spectrum in M13mp2 lacZα. Mutation spectra were determined by sequencing 135 (WT pol α) and 137 (L868F pol α) mutant plaques. The M13mp2 DNA sequence is shown. Base substitutions (A) (more ...)
The overall mutation rates and the error specificities, but not the base substitution specificities, of pol α L868F and translesion DNA polymerase of pol η are remarkably similar (34
). Interestingly, WT pol η has a Phe at the position corresponding to L868 in pol α and Phe is conserved for Y family DNA polymerases whereas Leu/Ile is conserved in family A/B polymerases (Fig. ).
Translesion DNA synthesis activity.
Y family DNA polymerases are characterized by low-fidelity DNA synthesis and efficient bypass of template DNA damage (i.e., translesion DNA synthesis). This suggests that pol α Leu868 and pol η Phe34 in S. cerevisiae might influence both fidelity of replication and efficiency of lesion bypass. Thus, the lesion bypass efficiencies of F34L pol η and L868F pol α were compared on a DNA template with a CPD lesion. WT pol η synthesized DNA on damaged and undamaged template DNA with nearly equal levels of efficiency. In contrast, it seems that F34L pol η was less efficient on damaged than on undamaged template DNA (Fig. ). The opposite effect was observed for WT and L868F pol α: the mutant bypassed the CPD lesion more efficiently than the WT. In addition, the mutant bypassed an abasic site and 6-4 photoproduct with higher efficiency than the WT. Even at 10-fold excess, WT pol α incorporates nucleotides extremely poorly compared to the results seen with the 3′ T of CPD or 6-4 photoproduct (Fig. ). These results show that pol α L868F has an increased bypass ability and a different lesion bypass specificity compared to WT pol η, which efficiently bypasses the CPD lesion but not the 6-4 photoproduct or abasic site.
FIG. 4. Translesion synthesis by pol η and α. Sodium dodecyl sulfate gel electrophoresis profiles for each enzyme preparation are shown in panels A (S. cerevisiae pol η WT and F34L), C (S. cerevisiae pol α WT and L868F), and E (more ...)
To quantify how well mutant and WT polymerases utilize the CPD template, single-nucleotide-incorporation kinetics investigations were carried out. F34L pol η incorporated dAMP 770-fold less efficiently than WT pol η at an undamaged T and 3,900-fold less efficiently than the WT at the 3′ T of a CPD (Table ). Thus, in pol η the mutation is primarily associated with the activity reduction, although F34L pol η discriminated between CPD and undamaged DNA 5.1-fold more efficiently than WT pol η, and it discriminated dGMP from dAMP 3.1-fold better at template T (Table ).
Fidelity of WT and mutant pol η on TT dimer by steady-state kinetics
The apparent kcat/Km values for L868F pol α were 30- and 26,000-fold higher at the undamaged T and 3′ T of the CPD, respectively, than those seen for WT pol α (Table ). Therefore, the discrimination factors at a CPD differ by 870-fold between L868F and WT pol α. In pol α, the L868F mutation dramatically increases the efficiency of nucleotide incorporation at the CPD lesion but has relatively little effect on polymerase activity on undamaged DNA.
Fidelity of WT and mutant pol α on TT dimer and abasic sites by steady-state kinetics
Kinetic analysis was also carried out for L868F pol α at other damaged and undamaged template sites. L868F pol α incorporated dAMP with similar levels of efficiency at the 5′ T and 3′ T of CPD (Table and Table ). The 5′-adjacent template C neighboring the CPD (designated CPD + 1) was utilized as efficiently as other undamaged template residues (Table ). Surprisingly, L868F incorporated dAMP opposite an abasic site or an undamaged template nucleotide with similar levels of efficiency (Table ). Furthermore, L868F pol α incorporated the pyrene nucleotide dPTP (33
) at the 3′ T of CPD as efficiently as it incorporated dAMP at an undamaged T (Table ).
Efficiency of S. cerevisiae pol α L868F at replicating through TT dimer and abasic sites by steady-state kinetics
pol α Leu868 is functionally and evolutionarily conserved.
Because the Leu/Ile residue is found in replicative DNA polymerases, it seems likely that it would be functionally and evolutionarily conserved. This idea is supported by the characteristics of WT and L864F human pol α. Primer extension analysis showed that human L864F pol α incorporated dAMP at the 3′ T of CPD 77-fold more efficiently than the WT (Fig. and Table ). The mutation frequency of human L864F pol α was 65-fold higher than that seen with the WT (Table ). Sequencing data showed that the overall error rate for human L864F was 0.012, or 1 in 80 nucleotides, which was 180-fold higher than seen with the WT (data not shown). Thus, WT and mutant human pol α are similar to their S. cerevisiae counterparts, strongly suggesting that Leu868/864 has a conserved function in yeast and human pol α. Like yeast pol η, the Leu mutant of human DNA pol η also decreased the translesion activity (data not shown).
Phenotype of Leu868 mutant pol α.
The in vitro data discussed above suggest that Leu868 in S. cerevisiae pol α is, at least in a part, a determinant of polymerase fidelity and that this residue might have played a role in the evolution of family B and Y DNA polymerases. If this hypothesis is correct, predictable phenotypic effects are to be expected in mutant yeast strains. This idea was tested by replacing the WT chromosomal pol α allele with an allele expressing L868F, L868M, or L868W pol α. The replication fidelity of these strains was measured using trp1 and his7 reversion assays and a CAN1 forward mutation assay (Table ). The trp1+, his7+, and CAN1r mutation rates in pol1-L868F yeast cells were 15-, 7.1-, and 8.1-fold higher, respectively, than those seen with the WT (Table ). In contrast, pol1-L868M and pol1-L868W yeast strains had trp1+ and his7+ reversion rates similar to those of the WT yeast and pol1-L868M had a moderate increase in the CAN1r forward mutation rate (2.9-fold higher than that of the WT). The base substitution specificity of these yeast strains was examined by limited sequencing of the trp1 gene in the revertants. In WT yeast, 82% of the revertants carried the original amber codon, indicating that the amber codon was suppressed by some other mechanism (e.g., mutation at distal site like a suppressor tRNA). The remaining revertants in WT yeast had mutations at the target codon including 27% transitions and 73% transversions (Table ). In pol1-L868F cells, 17% of the revertants retained the amber codon and the rest contained 8% transitions and 92% transversions at the target codon. In pol1-L868F yeast, the overall reversion rate at trp1-289 was 71 ×10−8, which is 42-fold higher than the trp1-289 reversion rate in WT yeast (1.7 × 10−8). At the can1 locus in WT and pol1-L868F cells, transitions were more frequent than transversions (Table ). Interestingly, N→T substitutions, which are characteristic of L868F pol α, also increased in the mutant strain.
Mutation rate of S. cerevisiae strains that carry either WT or mutant pol1a
Types of base substitutions observed with S. cerevisiae WT and pol1-L868F in trp1 reversion assay
Types of errors by S. cerevisiae WT and pol1-L868F in CAN1r forward mutation assaya
MMR recognition and repair of DNA synthesis errors introduced by L868M/W pol α.
Mutagenesis was examined in MMR-defective strains carrying L868M/W pol α. Targeted disruptions were introduced into MSH2, MSH3, or MSH6 in WT and mutant pol α strains, and the trp1 reversion rate was measured (Table ). In the double mutants msh2::pol1“msh2:: pol1-L868M/W and msh6::pol1-L868M/W, mutation rates increased synergistically. In contrast, disruption of msh3 did not significantly increase the mutation rate of pol1-L868M, pol1-L868W, or pol1-L868F. Similar results were observed using the CAN1 forward mutation assay (data not shown). These results suggest that DNA synthesis errors introduced by L868 pol α are recognized and repaired by MMR.