Characterization of G4 DNA formation in mitochondrial DNA and their potential role in mitochondrial genome instability

Abstract

Mitochondria possess their own genome which can be replicated independently of nuclear DNA. Mitochondria being the powerhouse of the cell produce reactive oxygen species, due to which the mitochondrial genome is frequently exposed to oxidative damage. Previous studies have demonstrated an association of mitochondrial deletions to aging and human disorders. Many of these deletions were present adjacent to non-B DNA structures. Thus, we investigate noncanonical structures associated with instability in mitochondrial genome. In silico studies revealed the presence of > 100 G-quadruplex motifs (of which 5 have the potential to form 3-plate G4 DNA), 23 inverted repeats, and 3 mirror repeats in the mitochondrial DNA (mtDNA). Further analysis revealed that among the deletion breakpoints from patients with mitochondrial disorders, majority are located at G4 DNA motifs. Interestingly, ~50% of the deletions were at base-pair positions 8271-8281, ~35% were due to deletion at 12362-12384, and ~12% due to deletion at 15516-15545. Formation of 3-plate G-quadruplex DNA structures at mitochondrial fragile regions was characterized using electromobility shift assay, circular dichroism (CD), and Taq polymerase stop assay. All 5 regions could fold into both intramolecular and intermolecular G-quadruplex structures in a KCl-dependent manner. G4 DNA formation was in parallel orientation, which was abolished in the presence of LiCl. The formation of G4 DNA affected both replication and transcription. Finally, immunolocalization of BG4 with MitoTracker confirmed the formation of G-quadruplex in mitochondrial genome. Thus, we characterize the formation of 5 different G-quadruplex structures in human mitochondrial region, which may contribute toward formation of mitochondrial deletions.

The Cellular Functions and Molecular Mechanisms of G-Quadruplex Unwinding Helicases in Humans

“…G4 DNA in mitochondria need to be unwound to maintain mitochondrial genome stability ( Bharti et al, 2014 ; Micol et al, 2019 ; Dahal et al, 2021 ), and G4 RNAs also participate in mitochondrial RNA metabolism ( Pietras et al, 2018 ). There are five helicases located in human mitochondria, including TWINKLE, SUV3, PIF1, DNA2, and RECQ4 ( Peter and Falkenberg, 2020 ).…”

Unleashing a novel function of Endonuclease G in mitochondrial genome instability

“…Previously, different non-B DNA motifs, including G4 DNA motifs have been reported in the mitochondrial genome ( Dahal et al, 2022 ; Damas et al, 2012 ; Dong et al, 2014 ). It was also suggested that alternate forms of DNA may play a role in the generation of deletions in mitochondria ( Dahal et al, 2022 ; Damas et al, 2012 ; Dong et al, 2014 ).…”

“…Previously, different non-B DNA motifs, including G4 DNA motifs have been reported in the mitochondrial genome ( Dahal et al, 2022 ; Damas et al, 2012 ; Dong et al, 2014 ). It was also suggested that alternate forms of DNA may play a role in the generation of deletions in mitochondria ( Dahal et al, 2022 ; Damas et al, 2012 ; Dong et al, 2014 ). In silico studies along with biochemical analyses revealed that 5 classical G4 DNA motifs are present in the mitochondrial genome, which could fold into G4 DNA ( Cer et al, 2013 ; Dahal et al, 2022 ; Figure 1—figure supplement 1A ).…”

“…It was also suggested that alternate forms of DNA may play a role in the generation of deletions in mitochondria ( Dahal et al, 2022 ; Damas et al, 2012 ; Dong et al, 2014 ). In silico studies along with biochemical analyses revealed that 5 classical G4 DNA motifs are present in the mitochondrial genome, which could fold into G4 DNA ( Cer et al, 2013 ; Dahal et al, 2022 ; Figure 1—figure supplement 1A ). Since ‘9 bp deletion’ (8271–8281) is the most frequent mitochondrial deletion and occurs next to one of the 5 G4 motifs with the coordinate position 8252–8295 (designated as mitochondrial Region I) ( Figure 1A and B ; Dahal et al, 2022 ), we have investigated the putative role of G4 DNA structure in DNA breakage and formation of the mtDNA deletions.…”

G‐quadruplexes in the mitochondrial genome – a cause for instability

“…In this issue, Dahal et al performed detailed in silico analysis using structure‐prediction tools to identify up to 35 non‐B DNA motifs in the mitochondrial genome [8]. These included G4 motifs, mirror repeats (for triplexes), inverted repeats (for cruciform DNA), Z DNA motif and A‐phased repeats.…”