Discovery of a major D-loop replication origin reveals two modes of human mtDNA synthesis.

Mammalian mitochondrial DNA (mtDNA) replication has long been considered to occur by asymmetric synthesis of the two strands, starting at the multiple origins of the strand-displacement loop (D-loop). We report the discovery of a major replication origin at position 57 in the D-loop of several human cell lines (HeLa, A549, and 143B.TK-) and immortalized lymphocytes. The nascent chains starting at this origin, in contrast to those initiated at the previously described origins, do not terminate prematurely at the 3' end of the D-loop but proceed well beyond this control point, behaving as "true" replicating strands. This origin is mainly responsible for mtDNA maintenance under steady-state conditions, whereas mtDNA synthesis from the formerly identified D-loop origins may be more important for recovery after mtDNA depletion and for accelerating mtDNA replication in response to physiological demands.