Asymmetric Context-Dependent Mutation Patterns Revealed through Mutation-Accumulation Experiments

Abstract

Despite the general assumption that site-specific mutation rates are independent of the local sequence context, a growing body of evidence suggests otherwise. To further examine context-dependent patterns of mutation, we amassed 5,645 spontaneous mutations in wild- type (WT) and mismatch-repair deficient (MMR(-)) mutation-accumulation (MA) lines of the gram-positive model organism Bacillus subtilis. We then analyzed>7,500 spontaneous base-substitution mutations across B. subtilis, Escherichia coli, and Mesoplasma florum WT and MMR(-) MA lines, finding a context-dependent mutation pattern that is asymmetric around the origin of replication. Different neighboring nucleotides can alter site-specific mutation rates by as much as 75-fold, with sites neighboring G:C base pairs or dimers involving alternating pyrimidine-purine and purine-pyrimidine nucleotides having significantly elevated mutation rates. The influence of context-dependent mutation on genome architecture is strongest in M. florum, consistent with the reduced efficiency of selection in organisms with low effective population size. If not properly accounted for, the disparities arising from patterns of context-dependent mutation can significantly influence interpretations of positive and purifying selection.

Keywords: Bacillus subtilis; context-dependent mutation; mismatch repair; mutation rate.

Fig. 1.

Rate and spectrum of base-substitution mutations in WT and MMR^– Bacillus subtilis MA lines. A–B. Distribution of base substitutions in 50 WT and 19 MMR^– B. subtilis MA lines. From the outer ring to inner ring scaled to genome size: significantly elevated (1 kb blocks that are >2 standard deviations from the genome-wide mean) gene density (gray), G/C content (blue), and A/T content (red), position of each base substitution in each MA line (black dots, with each circle representing the genome of an individual MA line), base-substitution density in 25 kb blocks (red >orange >yellow). When applicable, color intensity scales with increasing density for Circos plot (Krzywinski et al. 2009). C. Conditional (normalized for the number of considered G:C and A:T sites for each base-substitution mutation type) base-substitution mutation rates for WT and mutS^– B. subtilis MA lines. Error bars indicate the SEM. D. Conditional base-substitution mutation rates per site per generation of a subset of sites with varying flanking nucleotides in B. subtilis MMR^– MA lines. Standard DNA ambiguity codes are shown: N, any base (A|C|G|T); X, any base-substitution mutation (A|C|G|T); R, purine (A|G); Y, pyrimidine (C|T); S, strongly pairing base (C|G); W, weakly pairing base (A|T). Error bar indicates 95% confidence interval for each class.

Fig. 2.

Bilaterally symmetrical context-dependent mutation patterns in Bacillus subtilis MMR^– MA lines. A. Bidirectional fork at ORI displaying a 5′A-[G→X]-C3′ triplet in the leading-strand template. B. Heatmap of conditional base-substitution mutation rate (µ_x) of 64 possible triplet combinations in the left and right replichores, corresponding to the 5′ nucleotide, original nucleotide, and 3′ nucleotide with respect to the leading-strand template (Pearson’s correlation of the same triplet in each replichore, r = 0.98, P = 2.20 × 10⁻¹⁶, df = 62).

Fig. 3.

Bacillus subtilis MMR^–context-dependent mutation patterns compared with WT and other organisms. A. Log–log plot showing the correlation between context-dependent mutation rates for identical triplets in MMR^– and WT B. subtilis MA lines (Pearson’s correlation, r = 0.75, P = 9.28 × 10⁻¹¹, df = 62). B. Log-log plot displaying the relationship between the context-dependent mutation rates of identically synthesized triplets in the left and right replichores of MMR^– MA lines of Mesoplasma florum, B. subtilis, and Escherichia coli. The joint linear regression with equation log₁₀y = 0.87–0.99log₁₀x includes all points (r² = 0.77, P < 1 × 10⁻⁶, df = 162).

Fig. 4.

Observed correlation between existing genome-wide triplet count and expected genome-wide triplet usage at context-dependent mutation equilibrium in Mesoplasma florum. Log–log plot showing the relationship of the current genome-wide count of the 64 possible nucleotide triplets against the genome-wide count of the 64 possible nucleotide triplets at context-dependent mutation equilibrium in M. florum. Linear regression with equation log₁₀y = 2.80 + 0.37log₁₀x (r² = 0.90, P < 1 × 10⁻⁶, df = 62).