Fluorescence Imaging of Mitochondrial DNA Base Excision Repair Reveals Dynamics of Oxidative Stress Responses

Abstract

Mitochondrial function in cells declines with aging and with neurodegeneration, due in large part to accumulated mutations in mitochondrial DNA (mtDNA) that arise from deficient DNA repair. However, measuring this repair activity is challenging. We employ a molecular approach for visualizing mitochondrial base excision repair (BER) activity in situ by use of a fluorescent probe (UBER) that reacts rapidly with AP sites resulting from BER activity. Administering the probe to cultured cells revealed signals that were localized to mitochondria, enabling selective observation of mtDNA BER intermediates. The probe showed elevated DNA repair activity under oxidative stress, and responded to suppression of glycosylase activity. Furthermore, the probe illuminated the time lag between the initiation of oxidative stress and the initial step of BER. Absence of MTH1 in cells resulted in elevated demand for BER activity upon extended oxidative stress, while the absence of OGG1 activity limited glycosylation capacity.

Efficient DNA fluorescence labeling via base excision trapping

“…Two unusual features of the reaction of CCVJ-1 with AP sites in DNA are (a) an extremely rapid reaction rate, with rate constants up to ∼400 M −1 sec −1 at neutral pH, and (b) a high degree of light-up of the rotor dye, which is dark in solution but gives up to 500-fold enhancement upon covalent binding with AP sites in DNA. These features enabled its use in imaging DNA repair in cells and tissues; it was found that the dye in DNA is highly photostable and can be observed with common fluorescein filter sets, and can also be imaged by two-photon excitation 10 .…”

“…Here, we report an in situ DNA labeling strategy for oligonucleotides as well as dsDNA that makes use of aldehyde-reactive rotor dyes, also known as universal base excision reporters (UBER) 9 , 10 , to trap AP sites resulting from excision of deaminated DNA bases. This methodology includes (i) chemical or enzymatic synthesis of a DNA strand containing one or more deaminated bases, (ii) base excision repair (BER) in which a bacterial glycosylase excises the deaminated base(s), generating reactive AP sites, and (iii) oxime bond formation between the AP site and light-up UBER dyes (Fig.…”

Dynamic features of human mitochondrial DNA maintenance and transcription

“…Thus, despite recent reports showing that BER deficiency does not lead to increased mutation load in mtDNA or mtRNA in mice ( Kauppila et al, 2018 ), the fact that BER, as well as the nucleotide pool sanitizing enzymes MTH1 and deoxyuridine nucleotidohydrolase (dUTPase) that eliminates dUTP, exist in mitochondria indicate the importance of preventing and repairing base lesions in mtDNA ( Kang et al, 1995 ; Ladner and Caradonna, 1997 ; Ichikawa et al, 2008 ; Jun et al, 2022 ).…”

“…In a recent study, CRISPR-Cas9 based gene editing was used to generate cells lacking DNA glycosylase OGG1 that removes 8-oxoG from DNA, or MTH1 that hydrolyses 8-oxo-dGTP to 8-oxo-dGMP to prevent its insertion into DNA. In vivo mitochondrial BER activity was measured in OGG1-and MTH1-deficient cells using a cell-and mitochondrial-permeable fluorescent probe that reacts with AP sites in mtDNA ( Jun et al, 2022 ). Following exposure of the cells to an exogenous oxidating compound, mitochondrial BER was greatly elevated in the MTH1 KO cell line demonstrating the importance of sanitizing 8-oxoG precursors in mitochondria.…”

“…Following exposure of the cells to an exogenous oxidating compound, mitochondrial BER was greatly elevated in the MTH1 KO cell line demonstrating the importance of sanitizing 8-oxoG precursors in mitochondria. The OGG1 KO cells showed lower BER activity than both MTH1 KO cells and the wild-type control cells resulting in the accumulation of 8-oxoG lesions in mtDNA ( Jun et al, 2022 ).…”