Beyond translesion synthesis: polymerase κ fidelity as a potential determinant of microsatellite stability

Microsatellite DNA synthesis represents a significant component of human genome replication that must occur faithfully. However, yeast replicative DNA polymerases do not possess high fidelity for microsatellite synthesis. We hypothesized that the structural features of Y-family polymerases that facilitate accurate translesion synthesis may promote accurate microsatellite synthesis. We compared human polymerases κ (Pol κ) and η (Pol η) fidelities to that of replicative human polymerase δ holoenzyme (Pol δ4), using the in vitro HSV-tk assay. Relative polymerase accuracy for insertion/deletion (indel) errors within 2–3 unit repeats internal to the HSV-tk gene concurred with the literature: Pol δ4 >> Pol κ or Pol η. In contrast, relative polymerase accuracy for unit-based indel errors within [GT]10 and [TC]11 microsatellites was: Pol κ ≥ Pol δ4 > Pol η. The magnitude of difference was greatest between Pols κ and δ4 with the [GT] template. Biochemically, Pol κ displayed less synthesis termination within the [GT] allele than did Pol δ4. In dual polymerase reactions, Pol κ competed with either a stalled or moving Pol δ4, thereby reducing termination. Our results challenge the ideology that pol κ is error prone, and suggest that DNA polymerases with complementary biochemical properties can function cooperatively at repetitive sequences

What Is a Microsatellite: A Computational and Experimental Definition Based upon Repeat Mutational Behavior at A/T and GT/AC Repeats

Microsatellites are abundant in eukaryotic genomes and have high rates of strand slippage-induced repeat number alterations. They are popular genetic markers, and their mutations are associated with numerous neurological diseases. However, the minimal number of repeats required to constitute a microsatellite has been debated, and a definition of a microsatellite that considers its mutational behavior has been lacking. To define a microsatellite, we investigated slippage dynamics for a range of repeat sizes, utilizing two approaches. Computationally, we assessed length polymorphism at repeat loci in ten ENCODE regions resequenced in four human populations, assuming that the occurrence of polymorphism reflects strand slippage rates. Experimentally, we determined the in vitro DNA polymerase-mediated strand slippage error rates as a function of repeat number. In both approaches, we compared strand slippage rates at tandem repeats with the background slippage rates. We observed two distinct modes of mutational behavior. At small repeat numbers, slippage rates were low and indistinguishable from background measurements. A marked transition in mutability was observed as the repeat array lengthened, such that slippage rates at large repeat numbers were significantly higher than the background rates. For both mononucleotide and dinucleotide microsatellites studied, the transition length corresponded to a similar number of nucleotides (approximately 10). Thus, microsatellite threshold is determined not by the presence/absence of strand slippage at repeats but by an abrupt alteration in slippage rates relative to background. These findings have implications for understanding microsatellite mutagenesis, standardization of genome-wide microsatellite analyses, and predicting polymorphism levels of individual microsatellite loci

Variation in G-Quadruplex Sequence and Topology Differentially Impacts Human DNA Polymerase Fidelity

G-quadruplexes (G4s), a type of non-B DNA, play important roles in a wide range of molecular processes, including replication, transcription, and translation. Genome integrity relies on efficient and accurate DNA synthesis, and is compromised by various stressors, to which non-B DNA structures such as G4s can be particularly vulnerable. However, the impact of G4 structures on DNA polymerase fidelity is largely unknown. Using an in vitro forward mutation assay, we investigated the fidelity of human DNA polymerases delta (δ4, four-subunit), eta (η), and kappa (κ) during synthesis of G4 motifs representing those in the human genome. The motifs differ in sequence, topology, and stability, features that may affect DNA polymerase errors. Polymerase error rate hierarchy (δ4 \u3c κ \u3c η) is largely maintained during G4 synthesis. Importantly, we observed unique polymerase error signatures during synthesis of VEGF G4 motifs, stable G4s which form parallel topologies. These statistically significant errors occurred within, immediately flanking, and encompassing the G4 motif. For pol δ4, the errors were deletions, insertions and complex errors within the G4 or encompassing the G4 motif and surrounding sequence. For pol η, the errors occurred in 3\u27 sequences flanking the G4 motif. For pol κ, the errors were frameshift mutations within G-tracts of the G4. Because these error signatures were not observed during synthesis of an antiparallel G4 and, to a lesser extent, a hybrid G4, we suggest that G4 topology and/or stability could influence polymerase fidelity. Using in silico analyses, we show that most polymerase errors are predicted to have minimal effects on predicted G4 stability. Our results provide a unique view of G4s not previously elucidated, showing that G4 motif heterogeneity differentially influences polymerase fidelity within the motif and flanking sequences. Thus, our study advances the understanding of how DNA polymerase errors contribute to G4 mutagenesis

Microsatellite Interruptions Stabilize Primate Genomes and Exist as Population-Specific Single Nucleotide Polymorphisms within Individual Human Genomes

<div><p>Interruptions of microsatellite sequences impact genome evolution and can alter disease manifestation. However, human polymorphism levels at interrupted microsatellites (iMSs) are not known at a genome-wide scale, and the pathways for gaining interruptions are poorly understood. Using the 1000 Genomes Phase-1 variant call set, we interrogated mono-, di-, tri-, and tetranucleotide repeats up to 10 units in length. We detected ∼26,000–40,000 iMSs within each of four human population groups (African, European, East Asian, and American). We identified population-specific iMSs within exonic regions, and discovered that known disease-associated iMSs contain alleles present at differing frequencies among the populations. By analyzing longer microsatellites in primate genomes, we demonstrate that single interruptions result in a genome-wide average two- to six-fold reduction in microsatellite mutability, as compared with perfect microsatellites. Centrally located interruptions lowered mutability dramatically, by two to three orders of magnitude. Using a biochemical approach, we tested directly whether the mutability of a specific iMS is lower because of decreased DNA polymerase strand slippage errors. Modeling the adenomatous polyposis coli tumor suppressor gene sequence, we observed that a single base substitution interruption reduced strand slippage error rates five- to 50-fold, relative to a perfect repeat, during synthesis by DNA polymerases α, β, or η. Computationally, we demonstrate that iMSs arise primarily by base substitution mutations within individual human genomes. Our biochemical survey of human DNA polymerase α, β, δ, κ, and η error rates within certain microsatellites suggests that interruptions are created most frequently by low fidelity polymerases. Our combined computational and biochemical results demonstrate that iMSs are abundant in human genomes and are sources of population-specific genetic variation that may affect genome stability. The genome-wide identification of iMSs in human populations presented here has important implications for current models describing the impact of microsatellite polymorphisms on gene expression.</p></div

Sequence diversity created <i>in vitro</i> by human DNA polymerase η base substitution errors within perfect microsatellites.

<p>Bold, interrupting base(s).</p>a<p>Three independent occurrences.</p>b<p>A substitution occurred with a 1 unit deletion.</p>c<p>A substitution occurred with a 1 unit insertion.</p>d<p>A substitution occurred with a 2 unit deletion.</p

Pathways (substitutions, insertions, and deletions) driving the African population-specific interruptions.

<p>Repeats separated by (A) motif size, (B) repeat number, and (C) motif sequence for mono- and dinucleotides microsatellites.</p

DNA polymerase interruption mutagenesis within [GT]_n and [TC]_n dinucleotide microsatellite sequences.

<p>(A) Interruption Pol EFs at the [GT]₁₀, [GT]₁₉, and [TC]₁₁ alleles for B-family (pols α and δ), X-family (pol β) and Y-family (pols κ and η) DNA polymerases. Interruption Pol EFs were calculated from unpublished and published <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1004498#pgen.1004498-Kelkar1" target="_blank">[24]</a>, <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1004498#pgen.1004498-Baptiste1" target="_blank">[26]</a>, <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1004498#pgen.1004498-Eckert1" target="_blank">[45]</a>, <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1004498#pgen.1004498-Hile1" target="_blank">[46]</a>, <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1004498#pgen.1004498-Baptiste2" target="_blank">[48]</a>, <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1004498#pgen.1004498-Hile3" target="_blank">[94]</a> data by multiplying the proportion of interruption mutational events at each allele by the microsatellite Pol EF. Only detectable interruptions (ie, interruptions that produce a frameshift or a stop codon) were included in this analysis given that an event must be detectable to contribute toward the Pol EF. Less than symbol (<) indicates that no interruption events were found for pol α at the [GT]₁₀ allele; the interruption Pol EF is estimated to be <5.7×10⁻⁵. The Pol EF was not determined for Pol α or Pol η using the GT₁₉ template. (B) DNA polymerases utilize signature interruption mechanisms. Pie charts depict the proportion of mutational events generated by each possible interruption mechanism at [GT]_n and [TC]_n alleles. Graphs include both detectable and undetectable interruptions. Data used in the [GT]_n chart is a compilation of interruption events from pol β (N = 32) at [GT]₁₀, [GT]₁₃, and [GT]₁₉, pol κ (N = 36) at [GT]₁₀, [GT]₁₃, and [GT]₁₉, and pol η (N = 29) at [GT]₁₀. The [TC]_n chart includes events from pol β (N = 11) at [TC]₁₁ and [TC]₁₄, pol κ (N = 21) at [TC]₁₁ and [TC]₁₄, and pol η (N = 58) at [TC]₁₁. See Supplementary Figures S7 and S8 for complete representation of interruption mutations. (C) Detailed specificity of interruption events at [GT]_n and [TC]_n microsatellites. Columns in blue indicate the proportion of total interruptions that are single base deletions. Columns in red indicate the proportion that are single base insertions and columns in black/gray indicate the proportion that are base substitutions. Data used for this analysis is the same as that used in (B) for pols β, κ, and η. Data in combined column indicates the specificity obtained upon combining data from all three polymerases.</p

Effect of interruptions on microsatellite mutability in primate genomes.

<p>(A) Mutability of perfect (pure) microsatellites and that of microsatellites with one or two interruptions. (B) Mutability of perfect (pure) microsatellites and that of microsatellites with single interruptions that were located within the middle 25%, or in the fringe 25% (at either 5′ or 3′ end) of the microsatellite length. The number of repeats of a microsatellite was calculated by dividing the total length of the microsatellite, excepting the interrupting nucleotides, by the size of its repeating motif. At each repeat number the lines designate the 2.5th and 97.5th percentiles of empirical distributions that were obtained through bootstrap resampling. The repeats are binned based on their repeat number in the human genome (the reciprocal operation, when binning was based on repeat number in chimpanzee, did not change the results).</p