Regulation of mitochondrial D-loops by transcription factor A and single-stranded DNA-binding protein

During replication, mitochondrial DNA (mtDNA) takes on a triple‐stranded structure called a D‐loop. Although their physiological roles are not understood, D‐loops are implicated in replication and transcription of mtDNA. Little is known about the turnover of D‐loops. We investigated the effects of mitochondrial transcription factor A (TFAM) and single‐stranded DNA‐binding protein (mtSSB) on D‐loops. In human HeLa cells, TFAM and mtSSB are, respectively, 1700‐ and 3000‐fold more abundant than mtDNA. This level of TFAM is two orders of magnitude higher than reported previously and is sufficient to wrap human mtDNA entirely. TFAM resolves D‐loops in vitro if added in similar stoichiometries. mtSSB inhibits the resolution of mtDNA by TFAM but enhances resolution by RecG, a junction‐specific helicase from Escherichia coli . Hence, mtSSB functions in both stabilization and resolution. We propose that TFAM and mtSSB are cooperatively involved in stabilizing D‐loops and in the maintenance of mtDNA.