Leonie Wenson: Utilising DNA Modifying Enzymes for Method Development in Molecular Biology

Abstract

Method development plays a critical role in advancing molecular biology by enabling the detection, visualization, and interpretation of complex cellular processes. This dissertation focused on the development and optimization of methods based on DNA modifying enzymes to investigate DNA damage and protein–protein interactions—key mechanisms in genomic integrity, stress response, and gene regulation.

The first part of the work involved the development of Polymerase-Assisted DNA Damage Analysis (PADDA), a method combining the comet assay with enzymatic labelling to distinctively detect DNA single-strand breaks (SSBs) and double-strand breaks (DSBs) with fluorescence microscopy.

For a genome-wide detection of SSBs, a novel sequencing-based method—Sequence-Templated Erroneous End-Labelling sequencing (STEEL-seq) was developed. The method is based on an engineered, artificial DNA polymerase, Sloppymerase. Its highly error-prone activity allows for DNA synthesis in absence of a specific nucleotide (e.g. dATP), creating unique patterns of mismatches directly downstream of an SSB. These mismatches can be detected after DNA sequencing analysis and give information about bona fide SSBs. The method was validated using multiple sequencing platforms, revealing enrichment of SSBs at promoter regions of actively transcribed genes.

The final part of the work covers a new antibody-based proximity assay for the detection of endogenous protein-protein interactions - Enzyme-Activated Proximity of Oligonucleotides Sensing (EPOS). Across multiple cellular models, EPOS could produce robust results for the detection of PPIs with higher resolution, improved dynamic range and increased sensitivity compared with in situ proximity ligation assay.

Collectively, the methods developed during this project demonstrate the transformative potential of enzymatic tools in molecular biology. By enabling more precise and accessible analysis of DNA damage and protein interactions, these approaches provide valuable platforms for future research in genomics, cell biology, and biomedical science.