Logo: to the web site of Uppsala University

The ability to analyze whether DNA contains lesions is essential in identifying mutagenic substances. Currently, the detection of single-stranded DNA breaks (SSBs) lacks precision. To address this limitation, we develop a method for sequence-templated erroneous end-labelling sequencing (STEEL-seq), which enables the mapping of SSBs. The method requires a highly error-prone DNA polymerase, so we engineer a chimeric DNA polymerase, Sloppymerase, capable of replicating DNA in the absence of one nucleotide. Following the omission of a specific nucleotide (e.g., dATP) from the reaction mixture, Sloppymerase introduces mismatches directly downstream of SSBs at positions where deoxyadenosine should occur. This mismatch pattern, coupled with the retention of sequence information flanking these sites, ensures that the identified hits are bona fide SSBs. STEEL-seq is compatible with a variety of sequencing technologies, as demonstrated using Sanger, Illumina, PacBio, and Nanopore systems. Using STEEL-seq, we determine the SSB/base pair frequency in the human genome to range between 0.7 and 3.8 x 10-6 with an enrichment in active promoter regions.

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.

Introduction

Development of new methods is essential to make great leaps in science, opening up new avenues for research, but the process behind method development is seldom described.

Areas covered

Over the last twenty years we have been developing several new methods, such as in situ PLA, proxHCR, and MolBoolean, using oligonucleotide-conjugated antibodies to visualize protein-protein interactions. Herein, we describe the rationale behind the oligonucleotide systems of these methods. The main objective of this paper is to provide researchers with a description on how we thought when we designed those methods. We also describe in detail how the methods work and how one should interpret results.

Expert opinion

Understanding how the methods work is important in selecting an appropriate method for your experiments. We also hope that this paper may be an inspiration for young researchers to enter the field of method development. Seeing a problem is a motivation to develop a solution.

In the comet assay, tails are formed after single-cell gel electrophoresis if the cells have been exposed to genotoxic agents. These tails include a mixture of both DNA single-strand breaks (SSBs) and double-strand breaks (DSBs). However, these two types of strand breaks cannot be distinguished using comet assay protocols with conventional DNA stains. Since DSBs are more problematic for the cells, it would be useful if the SSBs and DSBs could be differentially identified in the same comet. In order to be able to distinguish between SSBs and DSBs, we designed a protocol for polymerase-assisted DNA damage analysis (PADDA) to be used in combination with the Flash comet protocol, or on fixed cells. By using DNA polymerase I to label SSBs and terminal deoxynucleotidyl transferase to label DSBs with fluorophore-labelled nucleotides. Herein, TK6-cells or HaCat cells were exposed to either hydrogen peroxide (H2O2), ionising radiation (X-rays) or DNA cutting enzymes, and then subjected to a comet protocol followed by PADDA. PADDA offers a wider detection range, unveiling previously undetected DNA strand breaks. Graphical Abstract