The in vitro fidelity of yeast DNA polymerase δ and polymerase ε holoenzymes during dinucleotide microsatellite DNA synthesis

Abstract

Elucidating the sources of genetic variation within microsatellite alleles has important implications for understanding the etiology of human diseases. Mismatch repair is a well described pathway for the suppression of microsatellite instability. However, the cellular polymerases responsible for generating microsatellite errors have not been fully described. We address this gap in knowledge by measuring the fidelity of recombinant yeast polymerase δ (Pol δ) and ɛ (Pol ɛ) holoenzymes during synthesis of a [GT/CA] microsatellite. The in vitro HSV-tk forward assay was used to measure DNA polymerase errors generated during gap-filling of complementary GT(10) and CA(10)-containing substrates and ∼90 nucleotides of HSV-tk coding sequence surrounding the microsatellites. The observed mutant frequencies within the microsatellites were 4 to 30-fold higher than the observed mutant frequencies within the coding sequence. More specifically, the rate of Pol δ and Pol ɛ misalignment-based insertion/deletion errors within the microsatellites was ∼1000-fold higher than the rate of insertion/deletion errors within the HSV-tk gene. Although the most common microsatellite error was the deletion of a single repeat unit, ∼ 20% of errors were deletions of two or more units for both polymerases. The differences in fidelity for wild type enzymes and their exonuclease-deficient derivatives were ∼2-fold for unit-based microsatellite insertion/deletion errors. Interestingly, the exonucleases preferentially removed potentially stabilizing interruption errors within the microsatellites. Since Pol δ and Pol ɛ perform not only the bulk of DNA replication in eukaryotic cells but also are implicated in performing DNA synthesis associated with repair and recombination, these results indicate that microsatellite errors may be introduced into the genome during multiple DNA metabolic pathways.

Figure 1. Determination of complete gap-filling by replicative polymerases

(A) Schematic representation of a polyacrylamide gel-based assay for detection of complete gap-filling. Cartoon depicts the pSStu2 gapped substrate created by hybridizing a sense ssDNA template to an antisense large fragment. The ∼100 nt MluI to StuI region of the gapped heteroduplex is used as a template for in vitro DNA synthesis. Potential products of the polymerase reaction can be completely (left) or incompletely (right) filled heteroduplexes. To determine the extent of the polymerase reaction, the reaction products are digested with MluI and StuI and the fragments are 5′ end-labeled through an exchange reaction with [γ-³²P] ATP and T4 kinase. Complete reactions will give two products of 111nt (nascent strand) and 115nt (template strand), whereas incomplete reactions will result in products less than 111nt. Complete reactions performed using the complementary gapped substrate (pSAStu2) with produce the opposite pattern: a 111nt product from the template strand and a 115nt product from nascent strand. (B) Analyses of the extent of gap-filling by Pol δ forms using the pSStu2 (GT₁₀) gapped substrate. Top panel: Cartoon depicting the gapped heteroduplex substrate. Middle panel: Agarose gel (0.6%) analysis of pol δ WT and Pol δ Exo- reaction products at varying enzyme:DNA molar ratios. Complete gap-filling reaction products will migrate at the position of the nicked substrate marker (N); G, unfilled gapped heteroduplex (starting substrate). Bottom panel: Polyacrylamide gel (6%) analysis of the same Pol δ reaction products after restriction enzyme digestion and [γ-³²P] end-labelling of DNA products. Arrow labeled T indicates the DNA band corresponding to the template strand. Arrow labeled N indicates the DNA band corresponding to the nascent strand. An identical amount of unfilled gapped substrate was either digested with MluI and StuI (+) or untreated (-), and analyzed as controls. M, 93nt and 97nt markers; 10nt ladder, 5′ end-labeled ladder. (C) Analyses similar to those shown in (B) of the extent of gap-filling by Pol δ forms using the pSAStu2 (CA₁₀) gapped substrate. Examples of incomplete reactions as shown by both the agarose and radioactive assays are shown in lanes indicated with asterisks (10:1 and ∼10:1 pol δ Exo- reactions). Fragments as short as 10 nt can be observed in the incomplete reactions with shorter electrophoresis times (data not shown).

Figure 2. The high fidelity of WT replicative polymerases is not maintained in dinucleotide microsatellite sequences

Gap-filling DNA synthesis reactions were performed using either (A). Polδ WT, or (B). Polε WT and the pSStu2/pSAStu2 complementary DNA substrates. The Pol MF_est was calculated from the HSV-tk mutant frequency, using the data presented in Table 2: Pol MF_est = [(HSV-tk MF of the indicated polymerase reaction) – (unfilled gap MF)] for each region. Graphs compare the Pol MF_est within the HSV-tk coding sequence (Open bars) to that within the [GT]₁₀ or [CA]₁₀ microsatellite sequences (solid bars) for each substrate.

Figure 3. The 3′ to 5′ exonuclease activity contributes little to the correction of replicative polymerase indel errors within microsatellite sequences

Gap-filling DNA synthesis reactions were performed using exonuclease-deficient and proficient forms of (A). Pol δ, or (B). Pol ε and pSStu2/pSAStu2 DNA substrates. The Pol MF_est values specifically for unit-based Indel errors within each microsatellite sequence were calculated using the frequency data in Table 2 and the proportion data in Table 3. Graphs compare the Pol MF_est for wild-type (open bars) and exonuclease deficient (solid bars) enzymes.