Every microsatellite is different: Intrinsic DNA features dictate mutagenesis of common microsatellites present in the human genome

Abstract

Microsatellite sequences are ubiquitous in the human genome and are important regulators of genome function. Here, we examine the mutational mechanisms governing the stability of highly abundant mono-, di-, and tetranucleotide microsatellites. Microsatellite mutation rate estimates from pedigree analyses and experimental models range from a low of approximately 10(-6) to a high of approximately 10(-2) mutations per locus per generation. The vast majority of observed mutational variation can be attributed to features intrinsic to the allele itself, including motif size, length, and sequence composition. A greater than linear relationship between motif length and mutagenesis has been observed in several model systems. Motif sequence differences contribute up to 10-fold to the variation observed in human cell mutation rates. The major mechanism of microsatellite mutagenesis is strand slippage during DNA synthesis. DNA polymerases produce errors within microsatellites at a frequency that is 10- to 100-fold higher than the frequency of frameshifts in coding sequences. Motif sequence significantly affects both polymerase error rate and specificity, resulting in strand biases within complementary microsatellites. Importantly, polymerase errors within microsatellites include base substitutions, deletions, and complex mutations, all of which produced interrupted alleles from pure microsatellites. Postreplication mismatch repair efficiency is affected by microsatellite motif size and sequence, also contributing to the observed variation in microsatellite mutagenesis. Inhibition of DNA synthesis within common microsatellites is highly sequence-dependent, and is positively correlated with the production of errors. DNA secondary structure within common microsatellites can account for some DNA polymerase pause sites, and may be an important factor influencing mutational specificity.

Figure 1. Quantitative effects of intrinsic microsatellite features on human cell mutation rates

(A). Motif Size; (B). Number of repeat units per allele; (C). Motif Sequence. Arrows point to the microsatellite allele with the higher mutation rate. Values are the relative difference in median mutation rate. Numbers in bold indicate differences that are statistically significant (p≤0.03, Mann-Whitney test). Data are taken from Table 1.

Figure 2. Microsatellite mutagenesis by purified polymerases α-primase and β

(A). Frameshift error frequencies at 2-4 nucleotide repeated sequences within the HSV-tk coding region are compared to artificial microsatellite sequences for pol α-primase (filled bars) and pol β (open bars). The average polymerase error frequency for complementary strands at each template sequence is graphed. Asterisk, not determined. (B). Specificity of pol α-primase (filled bars) and pol β (open bars) within the indicated frameshift and microsatellite regions. The proportion of expansion errors, relative to the total number of insertion and deletion errors, at each site is graphed. Asterisk, not determined. (C). Strand bias of errors produced by pol α-primase and pol β within microsatellite sequences. Data are from Refs. [38,39].

Figure 3. Sources of mutational biases observed in microsatellite sequences

The known effects of intrinsic features (Figure 1) on the efficiency of each step in the DNA slipped strand mispairing model are indicated. A, motif size; B, number of repeat units; C, motif sequence. See text for details.