Abstract
BackgroundShort tandem repeats (STRs) contribute significantly to de novo mutagenesis, driving phenotypic diversity and genetic disease. Although highly diverse, their repetitive sequences induce DNA polymerase slippage and stalling, leading to length and sequence variation. However, current studies of DNA synthesis through STRs are restricted to a handful of selected sequences, limiting our broader understanding of their evolutionary behaviour and hampering the characterisation of the determinants of their abundance and stability in eukaryotic genomes.ResultsWe perform a comprehensive analysis of DNA synthesis at all STR permutations and interrogate the impact of STR sequence and secondary structure on their genomic representation and mutability. To do this, we developed a high-throughput primer extension assay that allows monitoring of the kinetics and fidelity of DNA synthesis through 20,000 sequences comprising all STR permutations in different lengths. By combining these measurements with population-scale genomic data, we show that the response of a model replicative DNA polymerase to variously structured DNA is sufficient to predict the complex genomic behaviour of STRs, including abundance and mutational constraints. We demonstrate that DNA polymerase stalling at DNA structures induces error-prone DNA synthesis, which constrains STR expansion.ConclusionsOur data support a model in which STR length in eukaryotic genomes results from a balance between expansion due to polymerase slippage at repeated DNA sequences and point mutations caused by error-prone DNA synthesis at DNA structures.
Highlights
Short tandem repeats (STRs) contribute significantly to de novo mutagenesis, driving phenotypic diversity and genetic disease
Pooled measurement of DNA polymerase stalling at designed structured DNA sequences To address how DNA secondary structures impede DNA synthesis, we designed a library of 20,000 sequences (Additional file 2) and devised a method for accurately measuring polymerase stalling at these sequences in a single experiment
We present in this study a high-throughput primer extension assay for measuring the kinetics of DNA synthesis and DNA polymerase stalling at all short tandem repeats (STRs) permutations of different lengths
Summary
Short tandem repeats (STRs) contribute significantly to de novo mutagenesis, driving phenotypic diversity and genetic disease. Highly diverse, their repetitive sequences induce DNA polymerase slippage and stalling, leading to length and sequence variation. STRs are ubiquitous in eukaryotic genomes, with ~ 4,500,000 loci covering up to 2.5% of the human genome [3]. They exhibit mutation rates that are orders of magnitude higher than for other variant types such as single nucleotide polymorphisms (SNP) or copy number variations [4, 5]. The mechanisms driving and constraining the evolution of STRs length are poorly understood
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