R-loop accumulation promotes transcription-associated mutagenesis in SETX-deficient cells
Brooks, Laura
Brooks, Laura
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Abstract
DNA mutations are the primary drivers of cancer, arising from endogenous and exogenous sources. My research focuses on transcription-associated mutagenesis (TAM) of human DNA, an understudied cancer etiology. Our lab hypothesizes that R-loops (RNA/DNA hybrids) promote transcription-associated large insertion and deletion mutations by contributing to double-strand break formation. To test this hypothesis, I generated two independent CRISPR-mediated knockouts of SETX, an RNA/DNA helicase that resolves R-loops. ?H2AX-foci and DRIP-qPCR assays found a significant increase in R-loop accumulation and double-strand break formation in knockout cells compared to wild-type controls. A reporter-based mutation rate and spectrum assay revealed an unexpectedly high mutation rate that prevented precise quantification. Subsequent sequencing of select mutants found numerous large deletions and templated insertions, consistent with our proposed mechanism. However, this elevated mutagenicity ultimately caused duplication of the reporter locus, limiting the use of this system for further experimentation. Together, these findings suggest that elevated R-loop accumulation in SETX-deficient cells produces a high transcription-associated mutagenic burden, motivating further investigation into the mechanistic role of R-loops in transcription-associated genomic instability.
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Date
1/1/2026
Student Status
Senior (Graduating in 2026)
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Poster
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Molecular Genetics
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College of Agriculture and Life Sciences
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Life Science