In human mitochondria the transcription machinery generates the RNA primers 
needed for initiation of DNA replication. A critical feature of the 
leading-strand origin of mitochondrial DNA replication is a CG-rich element 
denoted conserved sequence block II (CSB II). During transcription of CSB II, a 
G-quadruplex structure forms in the nascent RNA, which stimulates transcription 
termination and primer formation. Previous studies have shown that the newly 
synthesized primers form a stable and persistent RNA-DNA hybrid, a R-loop, near 
the leading-strand origin of DNA replication. We here demonstrate that the 
unusual behavior of the RNA primer is explained by the formation of a stable 
G-quadruplex structure, involving the CSB II region in both the nascent RNA and 
the non-template DNA strand. Based on our data, we suggest that G-quadruplex 
formation between nascent RNA and the non-template DNA strand may be a regulated 
event, which decides the fate of RNA primers and ultimately the rate of 
initiation of DNA synthesis in human mitochondria.