Figure 6

AtPrimPol synthetizes primers that are elongated by plant organellar DNA polymerases. (A) A coupled primase-DNA polymerase reaction containing 200 nM of AtPrimPol and 200 nM of AtPolIA or AtPolIB was setup. The reactions were incubated the presence of dNTPs and labeled with dGTP, using as template a ssDNA M13 vector, reactions were loaded in a 0.6% agarose gel. Both coupled reactions AtPrimPol-AtPolIA (lanes 5–9) and AtPrimPol-AtPoIlB (lanes 10–14) were able to replicate the ssDNA vector of 7.2 kbp at the lowest reaction time. Increasing the reaction time leads to an increase of size of the DNA product (up to 23 kbp) due to the strand-displacement activity of the POPs. AtPrimPol (lane 2), AtPolIA (lane 3) and AtPolIB (lane 3) on their own, were unable to synthetize the full-length DNA product at the highest reaction time (lanes 2–4). (B) M13 ssDNA was incubated with increasing concentrations (0, 620, 1250, 2500 y 5000 nM) of organellar single-stranded binding proteins (OSSBs) AtmtSSB1, AtOSB2 and AtWhy2 at room temperature during 30 min, afterwards a mixture of 200 nM of AtPrimPol and AtPoIlA was added and the reaction was incubated at 37 °C during 40 min. Reactions were loaded onto a 0.6% agarose gel. The reactions in presence of AtWhy2 (lanes 13–17), showed that this protein efficiently blocks or inhibit the replication from 620 nM where the half of product was reduced. In the AtOSB2′ s reactions (lanes 8–12) the fall of de product is more slowly, even at the highest concentration some product was produced. Whereas the reactions with AtmtSSB1 (lanes 3–7) showed that AtPrimPol-AtPolI were able to replicate the ssDNA vector while the concentration of AtmtSSB1 was increasing. Original data used to compose this figure is present in Fig. S12.