Detection and mutational consequences of embedded ribonucleotides

Abstract

Mutation is a fundamental driver of evolution. It occurs non-randomly throughout the genome, influenced by factors such as chromatin architecture, DNA replication, transcription and repair. In human populations an important, but poorly understood, subset of mutations are short insertions and deletions (indels), the formation of which has traditionally been ascribed to deficiencies in mismatch repair to correct polymerase slippage events. However in S. cerevisiae, the formation of short 2 to 5 base pair deletions has been shown to arise as a consequence of the activity of Topoisomerase 1 (Top1) on DNA-embedded ribonucleotides. In normal cells, such ribonucleotides are the most common aberrant non-canonical nucleotides. They occur stochastically throughout the genome, mainly as a result of polymerase misincorporation. In this thesis I detail the development of a novel, nanopore based methodology to detect ribonucleotides embedded in DNA at single nucleotide resolution. I also describe the design and implementation of a highly sensitive reporter construct in S.cerevisiae to detect the Top1 dependent deletion signature. Transferring this reporter to HeLa cells, I show that the same deletion signature is also present in human cells, and confirm this through the orthogonal analysis of a whole genome sequencing mutation accumulation experiment in RPE1 cells. I identify the same mutation signature in de novo mutations from human populations, showing similarities between this mutational process and a newly described COSMIC (Catalogue of Somatic Mutations in Cancer) indel signature. This work demonstrates the presence of a novel mutational process in human genomes, distinct to that caused by polymerase slippage and deficient mismatch repair activity. This process may be an important cause of short deletions in human cells, and has implications for our understanding of the generation of genetic diversity, the formation of de novo mutations, and the development of cancer.