Current Research Methods to Evaluate DNA Damage and Repair

TOPICAL COLLECTIONS

Guest Editor

Luiza Steffens Reinhardt

University of Newcastle

Dr. Luiza Steffens Reinhardt is a post-doctoral researcher at the University of Newcastle (Australia) working on the molecular alterations in cancers that lead to resistance to DNA-damaging therapies. Luiza received her PhD in Biosciences from the Federal University of Health Sciences of Porto Alegre in 2022 in the field of molecules with biotechnological potential and has submitted her thesis for a second doctorate degree in Molecular Genetics from the University of Newcastle in the field of p53 isoforms and their links to chemotherapy resistance in breast cancer. Luiza has acquired extensive experience with DNA damage signaling pathways, particularly with protein kinases involved in the DNA damage checkpoint activation during her previous studies under the supervision of Prof Dinara Moura at the Genetic Toxicology laboratory (UFCSPA).Her current research is particularly focused on targeting the p53 isoforms and using these variants as disease biomarkers. Her research under the supervision of A/Prof Kelly Avery-Kiejda on the role of the p53 isoforms in response to DNA-damaging therapies has underpinned novel mechanisms of disruption of the p53 canonical DNA damage response and has led to the identification of novel markers of breast cancer recurrence.

Collection Overview

Endogenous and exogenous genotoxic stressors can cause changes in DNA structure by introducing mismatched or oxidized bases, adducts, bulky lesions, DNA cross-linking, and single- and double-stranded breaks. As a result, more than one million individual DNA lesions may occur per cell each day. Cells activate various mechanisms in response to DNA damage, including sustaining, reversing, or repairing DNA lesions, or initiating cell death mechanisms in cases of irreparable damage. These mechanisms collectively form the DNA damage response, which encompasses a complex network of DNA damage sensors, signaling pathways, and DNA repair or cell death effectors.

An ineffective or dysregulated DNA damage response may result in genomic instability due to the accumulation of DNA mutations, which can contribute to inflammation and disease. To comprehensively study how cells respond to DNA damage and the presence of DNA lesions, it is crucial to employ appropriate methods for detecting DNA damage. This Methods Collection includes a variety of approaches for studying DNA damage and repair. Some examples of methods to be included in this collection are:

· Molecular strategies including quantitative, ligation-mediated, or immune-coupled PCR, expression analysis of signaling and repair markers (DNA repair pathway proteins, p53 family of proteins, phosphorylated histone 2AX), DNA repair reporter plasmids, and DNA fiber assay.

· Fluorescence strategies such as comet assays (neutral, alkaline, lesion-specific enzyme-based), TUNEL assay, FISH (bromodeoxyuridine-labeled DNA-comet, DNA breakage detection), Halo assay, FCM-Annexin V labeling, radioimmunoassay.

· Chemiluminescence strategies such as ELISA and immunohistochemical/logical assays.

· Analytical strategies to identify modified bases (UV-induced dimeric pyrimidine photoproducts or oxidative-damaged DNA) such as HPLC-MS, GS-MS, and electrochemical methods.