High-resolution mapping of abasic sites and pyrimidine modifications in DNA
The maintenance of genomic stability is critical for the growth and survival of cells. Cellular DNA is constantly subject to both endogenous and exogenous sources of damage, leading to the formation of damage products. Abasic sites occur when a nucleobase is lost from DNA by hydrolysis and can lead to mutations and genomic instability. This thesis focuses on the development and application of methodology to map the location of abasic sites in DNA by next-generation sequencing. Chemically, abasic sites are reactive entities due to the aldehyde moiety in the ring-open form of the deoxyribose. Many studies on abasic sites have focused on targeting this aldehyde, however, a major drawback to this approach is the cross-reactivity of nucleophilic probes with other aldehyde-containing modifications that naturally occur within genomic DNA. In this thesis, a chemical method was developed and demonstrated to allow the sequencing of abasic sites at single-nucleotide resolution by affinity enrichment. Crucially, this method was shown to selectively target abasic sites in the presence of other reactive sites in DNA. Glycosylase enzymes excise base modifications from DNA to generate an abasic site. By treating isolated DNA with a glycosylase in vitro, abasic site sequencing methodology can be widely applied to study a range of DNA base modifications. This approach was utilised to investigate the distribution of the modification 5-hydroxymethyluracil at single-nucleotide resolution in the DNA of trypanosomatids. This study provided proof-of-concept of the sequencing methodology in a genomic context and also revealed the genomic features and sequence motifs at which these sites accumulate. The distribution of endogenous abasic sites was also explored in the human genome. The genomic location of abasic sites was mapped following depletion of the key repair enzyme, APE1, where an increase was observed in the number of enriched loci compared to control cells. A relationship was also revealed between this form of DNA damage and coding and regulatory regions of the genome upon knockdown of the APE1 protein. The genomic location of the base modification uracil was studied in the mouse genome. This base has been implicated in a pathway proposed to regulate the epigenetic DNA marker, 5-methylcytosine during embryonic development. Sequencing of uracil was achieved by enzymatic conversion into abasic sites followed by genome-wide mapping to investigate the extent to which the proposed mechanism contributes towards epigenetic reprogramming. Overall, a versatile method has been developed that can reveal the location of endogenous abasic sites within DNA at single-nucleotide resolution, as well as abasic sites generated in vitro from base modifications. This method is useful for studies on both DNA damage and DNA base modifications.
DNA damage, abasic sites, epigenetics, 5-hydroxymethyluracil, uracil
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