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

Abstract

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.

Fig. 1. Quantification of TFAM, mtSSB and mtDNA. (A) The purified recombinant His-TFAM and HeLa cell lysates were run in the same gel. The amount of His-TFAM was adjusted to yield a signal intensity similar to that of endogenous TFAM in western blots. One microgram protein of the lysate corresponds to 1.7 × 10³ cells. (B) Western blots for quantification of mtSSB. Procedures are the same as in (A). The N-terminal His tag of His-mtSSB was removed with TEV protease (TEV-mtSSB). (C) Southern blots for quantification of mtDNA. Signals of the standard DNA pBCO2 are shown (lanes 3–5). In lane 2, endogenous mtDNA and an internal standard are shown. mtDNA used for the resolution assay is in lane 1. (D) Total DNA extracted from the indicated volume of P2 (1 mg protein/ml) was analyzed as in (C) (upper). His-TFAM, P2 mixed with His-TFAM and P2 alone were analyzed in western blots (lower). (E) P2 or DNA extracted from P2 was incubated with various concentrations of micrococcal nuclease (MNase) and then mtDNA was detected in Southern blots using a probe covering nucleotide position (np) 7441–8286. One unit is an activity producing one OD₂₆₀ of acid-soluble materials from calf thymus DNA at 37°C for 30 min.

Fig. 1. Quantification of TFAM, mtSSB and mtDNA. (A) The purified recombinant His-TFAM and HeLa cell lysates were run in the same gel. The amount of His-TFAM was adjusted to yield a signal intensity similar to that of endogenous TFAM in western blots. One microgram protein of the lysate corresponds to 1.7 × 10³ cells. (B) Western blots for quantification of mtSSB. Procedures are the same as in (A). The N-terminal His tag of His-mtSSB was removed with TEV protease (TEV-mtSSB). (C) Southern blots for quantification of mtDNA. Signals of the standard DNA pBCO2 are shown (lanes 3–5). In lane 2, endogenous mtDNA and an internal standard are shown. mtDNA used for the resolution assay is in lane 1. (D) Total DNA extracted from the indicated volume of P2 (1 mg protein/ml) was analyzed as in (C) (upper). His-TFAM, P2 mixed with His-TFAM and P2 alone were analyzed in western blots (lower). (E) P2 or DNA extracted from P2 was incubated with various concentrations of micrococcal nuclease (MNase) and then mtDNA was detected in Southern blots using a probe covering nucleotide position (np) 7441–8286. One unit is an activity producing one OD₂₆₀ of acid-soluble materials from calf thymus DNA at 37°C for 30 min.

Fig. 2. Effect of TFAM on D-loop structure. (A) mtDNA was incubated at 37°C for 30 min with various concentrations of TFAM (lanes 2–10). In lane 1, mtDNA was incubated at 90°C for 10 min. Each sample was analyzed in Southern blots. (B) Ethidium bromide staining of supercoiled plasmid pBR322. The plasmids were incubated with (lanes 3 and 4) or without (lanes 1 and 2) 8 µM TFAM at 37°C for 30 min, followed by incubation without (lanes 1 and 3) or with (lanes 2 and 4) proteinase K (PK) at 37°C for 15 min. In lane 5, plasmids were linearized with EcoRI.

Fig. 3. Effects of mtSSB on D-loop resolution. (A) pBR322 plasmids were incubated with 8 µM mtSSB (lane 2) and a vehicle (lane 1), electrophoresed on an agarose gel and then stained with ethidium bromide. mtDNA was preincubated with the indicated concentration of mtSSB, followed by incubation with 5 µM TFAM (B), 20 mM MPP⁺ (C), 5 µM TFAM and 10 mM MPP⁺ (D) and 200 nM of RecG (E).

Fig. 3. Effects of mtSSB on D-loop resolution. (A) pBR322 plasmids were incubated with 8 µM mtSSB (lane 2) and a vehicle (lane 1), electrophoresed on an agarose gel and then stained with ethidium bromide. mtDNA was preincubated with the indicated concentration of mtSSB, followed by incubation with 5 µM TFAM (B), 20 mM MPP⁺ (C), 5 µM TFAM and 10 mM MPP⁺ (D) and 200 nM of RecG (E).