This study is the first to investigate the in vitro
fidelity of replicative, eukaryotic holoenzymes within a tandem repeat array that can be defined as a microsatellite allele [3
]. Using the well-defined in vitro
HSV-tk polymerase fidelity assay [3
], high polymerase mutant frequencies (~10-3
) were measured during microsatellite DNA synthesis by the wild-type holoenzymes, which contrasts with the low mutant frequencies (~10-4
) measured during synthesis of a non-repetitive sequence (). Error correction by the respective proofreading exonucleases contributed little to the overall fidelity of the polymerases within the microsatellite sequences (). These data support the previous suggestion that microsatellites are “at-risk” sequences for variation within the eukaryotic genome [46
We investigated whether the high fidelity of replicative polymerases previously measured for base substitution and indel errors within genes would be mirrored in longer, tandem repeats (e.g., microsatellites). As clearly shown here, high fidelity is not maintained for either Pol δ or Pol ε within the [GT/CA]10
microsatellite. Directly comparing misalignment-based errors, the fidelity of Pol δ WT and Pol ε WT for unit-based indel errors created during synthesis of the microsatellite alleles is ~1000-fold lower than the fidelity for indel errors created during HSV-tk gene synthesis (). The results for Pol ε were unexpected, as this polymerase is among the most accurate of DNA polymerases in vitro
for single base indel errors [42
]. The addition of the replication accessory proteins RPA, RFC and PCNA to yeast Pol δ in vitro
reactions was previously shown not to alter the error rate for one nucleotide deletions [33
]. Likewise, the addition of RFC and PCNA to human Pol δ reactions does not alter the frequency of errors within the GT10
. [GT/CA] mutagenesis during genomic DNA replication will be the summation of polymerase errors on the [GT] strand plus errors on the [CA] strand. Assuming that Pol δ and Pol ε replicate complementary strands and that the polymerases display similar fidelity in vivo
, we estimate conservatively that the combined Pol δ + Pol ε error frequency during [GT+CA] synthesis is 2.5-5 × 10-3
(). This frequency corresponds to an expected mutation rate of 1 mutant per 200 – 400 [GT/CA] alleles per round of DNA replication.
Estimated microsatellite mutation frequencies due to wild-type replicative holoenzyme errors during DNA replication
Previously, the error rates of Pol δ WT and Pol ε WT during synthesis of seven consecutive thymines were determined using an in vitro
lacZ gap-filling assay, similar to the HSV-tk assay [44
]. Using those data, we calculated the error rate per repeat unit for Pol δ and Pol ε at the mononucleotide T allele versus
the GT dinucleotide allele (). This analysis suggests that the fidelity of Pol δ and Pol ε for mononucleotide microsatellite DNA synthesis may be even lower, as the polymerases created errors more often within the [T]7
allele than within the [GT]10
allele. However, the effects of repeat unit size (mono-, di- tetra) on replicative polymerase error rates must be tested directly in future studies using the same mutational assay.
Comparison of wild-type polymerase error rates within mononucleotide and dinucleotide tandem repeats
We also examined the importance of the intrinsic 3′ to 5′ exonuclease activity to microsatellite sequence variation. The frequency of unit-based microsatellite indel errors produced by exonuclease-deficient enzymes differed by less than 1.4-fold from that measured for the corresponding exonuclease-proficient enzymes, with the exception of Pol δ and the CA allele (). Interestingly, the exonucleases tend to preferentially remove interruption errors within the microsatellite alleles (). Such interruptions, if maintained over several rounds of DNA replication, would be expected to stabilize genomic microsatellites by breaking an allele into two, shorter tandem arrays that mutate at lower frequencies than the parent allele. Thus, the normally protective proofreading function may act instead to promote genome instability within microsatellite sequences. These in vitro
results are consistent with previous in vivo
studies showing that the proofreading activities of both Pol δ and Pol ε are inefficient at recognizing and repairing indel mutations in [GT/CA] repeats [29
]. The low proofreading efficiency within microsatellites may be due to stabilization of the misaligned intermediate over the entire length of the repeated array, resulting in minimal distortion of the DNA substrate. Structurally, the mere physical distance over which to incorporate a bulge of unpaired bases within a long repetitive sequence may result in the physical separation of the polymerase active site from the misalignment, such that the bulge is rendered invisible to the proofreading function.
Evolutionary models of microsatellite mutation have been developed for use in estimates of genetic distances between populations (reviewed in [2
]). The widely used stepwise mutation models for microsatellite mutation assume that insertions or deletions of a single unit occur at fixed rates as a function of allele length. A full 20% of the indel mutations created by the wild-type replicative polymerases within the dinucleotide alleles were deletions of two or more repeat units, as measured in the HSV-tk assay (). These observations suggest that Pol δ and Pol ε may be able to accommodate large loops of extrahelical nucleotides during extension synthesis. Alternatively, the multi-unit deletions may result from the simultaneous formation of multiple, single unit bulges within the long [GT/CA]10
repeated tract. Further investigation is required to elucidate the mechanistic underpinnings of the multi-unit repeat deletions. Regardless of mechanism, the incidence of multi-unit deletions within the [GT/CA] microsatellite should be taken into account in future mathematical models of microsatellite evolution.
Finally, small strand biases in replicative polymerase fidelity during [GT]10
microsatellite synthesis were observed (). This bias is intriguing, as a current model for genome replication is that Pol δ synthesizes the lagging strand template [40
] and Pol ε synthesizes the leading strand template [41
]. The in vitro
data presented here predict a strand bias in microsatellite mutability may exist in vivo
, depending on the position of the GT versus the CA dinucleotide sequence relative to the origin of DNA replication (). We plan to further investigate a microsatellite strand bias for DNA polymerase errors in vivo
, using a yeast reporter cassette adjacent to a well-defined origin of replication in a yeast strain with specialized Pol δ and Pol ε mutator alleles with wild type catalytic activity and strong mutational specificity [40
]. In yeast, differences in the specificity and efficiency of MMR correction have been observed among A10
, and G10
]. The results of this study support the hypothesis that these biases reflect, in part, the error specificities of the replicative polymerases that initiate the mutation.