Genetic evidence that both dNTP-stabilized and strand slippage mechanisms may dictate DNA polymerase errors within mononucleotide microsatellites |
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| Affiliation: | 1. Department of Radiation Oncology, Houston Methodist Research Institute, Houston, TX 77030, USA;2. Houston Methodist Neurological Institute, Houston, TX 77030, USA;3. Weill Medical College of Cornell University, New York, USA;1. Department of Radiation Oncology, Houston Methodist Research Institute, Houston, TX 77030, USA;2. Weill Medical College of Cornell University, New York, USA;1. University of Utah School of Medicine, Salt Lake City, UT 84132, United States;2. Oregon Institute of Occupational Health Sciences, Oregon Health & Science University, Portland, OR, 97239, United States;3. Nzumbe Inc., Portland, OR, 97201, United States;4. Department of Molecular and Medical Genetics, Oregon Health & Science University, Portland, OR, 97239, United States;1. Department of Microbiology and Immunology, University of Texas Medical Branch at Galveston, Galveston, TX 77555, USA;2. Sealy Center for Molecular Medicine, University of Texas Medical Branch at Galveston, Galveston, TX 77555, USA;3. Department of Biochemistry and Molecular Biology, University of Texas Medical Branch at Galveston, Galveston, TX 77555, USA;1. Department of Medicine and Duke Clinical Research Institute, Duke University School of Medicine, Durham, NC 27701, USA;2. Duke Forge, Duke University School of Medicine, Durham, NC 27701, USA;3. Department of Medicine, Stanford University, Stanford, CA, USA;4. Verily Life Sciences (Alphabet), South San Francisco, CA, USA |
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| Abstract: | Mononucleotide microsatellites are tandem repeats of a single base pair, abundant within coding exons and frequent sites of mutation in the human genome. Because the repeated unit is one base pair, multiple mechanisms of insertion/deletion (indel) mutagenesis are possible, including strand-slippage, dNTP-stabilized, and misincorportion-misalignment. Here, we examine the effects of polymerase identity (mammalian Pols α, β, κ, and η), template sequence, dNTP pool size, and reaction temperature on indel errors during in vitro synthesis of mononucleotide microsatellites. We utilized the ratio of insertion to deletion errors as a genetic indicator of mechanism. Strikingly, we observed a statistically significant bias toward deletion errors within mononucleotide repeats for the majority of the 28 DNA template and polymerase combinations examined, with notable exceptions based on sequence and polymerase identity. Using mutator forms of Pol β did not substantially alter the error specificity, suggesting that mispairing-misalignment mechanism is not a primary mechanism. Based on our results for mammalian DNA polymerases representing three structurally distinct families, we suggest that dNTP-stabilized mutagenesis may be an alternative mechanism for mononucleotide microsatellite indel mutation. The change from a predominantly dNTP-stabilized mechanism to a strand-slippage mechanism with increasing microsatellite length may account for the differential rates of tandem repeat mutation that are observed genome-wide. |
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| Keywords: | Microsatellite Mutation bias DNA polymerase fidelity DNA misalignment Strand slippage indel mutation Mononucleotide |
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