BACKGROUND: Mitochondrial DNA (mtDNA) deletions cause disease and accumulate 
during aging, yet our understanding of the molecular mechanisms underlying their 
formation remains rudimentary. Guanine-quadruplex (GQ) DNA structures are 
associated with nuclear DNA instability in cancer; recent evidence indicates 
they can also form in mitochondrial nucleic acids, suggesting that these non-B 
DNA structures could be associated with mtDNA deletions. Currently, the multiple 
types of GQ sequences and their association with human mtDNA stability are 
unknown.
RESULTS: Here, we show an association between human mtDNA deletion breakpoint 
locations (sites where DNA ends rejoin after deletion of a section) and 
sequences with G-quadruplex forming potential (QFP), and establish the ability 
of selected sequences to form GQ in vitro. QFP contain four runs of either two 
or three consecutive guanines (2G and 3G, respectively), and we identified four 
types of QFP for subsequent analysis: intrastrand 2G, intrastrand 3G, duplex 
derived interstrand (ddi) 2G, and ddi 3G QFP sequences. We analyzed the position 
of each motif set relative to either 5' or 3' unique mtDNA deletion breakpoints, 
and found that intrastrand QFP sequences, but not ddi QFP sequences, showed 
significant association with mtDNA deletion breakpoint locations. Moreover, a 
large proportion of these QFP sequences occur at smaller distances to 
breakpoints relative to distribution-matched controls. The positive association 
of 2G QFP sequences persisted when breakpoints were divided into clinical 
subgroups. We tested in vitro GQ formation of representative mtDNA sequences 
containing these 2G QFP sequences and detected robust GQ structures by UV-VIS 
and CD spectroscopy. Notably, the most frequent deletion breakpoints, including 
those of the "common deletion", are bounded by 2G QFP sequence motifs.
CONCLUSIONS: The potential for GQ to influence mitochondrial genome stability 
supports a high-priority investigation of these structures and their regulation 
in normal and pathological mitochondrial biology. These findings emphasize the 
potential importance of helicases that subsequently resolve GQ to maintain the 
stability of the mitochondrial genome.