Mitochondrial DNA deletions are prominent in human genetic disorders, cancer, 
and aging. It is thought that stalling of the mitochondrial replication 
machinery during DNA synthesis is a prominent source of mitochondrial genome 
instability; however, the precise molecular determinants of defective 
mitochondrial replication are not well understood. In this work, we performed a 
computational analysis of the human mitochondrial genome using the "Pattern 
Finder" G-quadruplex (G4) predictor algorithm to assess whether G4-forming 
sequences reside in close proximity (within 20 base pairs) to known 
mitochondrial DNA deletion breakpoints. We then used this information to map G4P 
sequences with deletions characteristic of representative mitochondrial genetic 
disorders and also those identified in various cancers and aging. Circular 
dichroism and UV spectral analysis demonstrated that mitochondrial G-rich 
sequences near deletion breakpoints prevalent in human disease form G-quadruplex 
DNA structures. A biochemical analysis of purified recombinant human Twinkle 
protein (gene product of c10orf2) showed that the mitochondrial replicative 
helicase inefficiently unwinds well characterized intermolecular and 
intramolecular G-quadruplex DNA substrates, as well as a unimolecular G4 
substrate derived from a mitochondrial sequence that nests a deletion breakpoint 
described in human renal cell carcinoma. Although G4 has been implicated in the 
initiation of mitochondrial DNA replication, our current findings suggest that 
mitochondrial G-quadruplexes are also likely to be a source of instability for 
the mitochondrial genome by perturbing the normal progression of the 
mitochondrial replication machinery, including DNA unwinding by Twinkle 
helicase.