Figure 4.

RPA stimulates both primase and polymerase activities of PrimPol in synergy with Polε. (a) Effect of RPA on the combined primase and polymerase activities of PrimPol in non-primed M13 ssDNA. PrimPol (200 nM) was incubated with the template M13 ssDNA 2 nM, 100 µM of dATP, dCTP, dTTP and GTP, 16 nM [α-³²P] dGTP in the presence of 5 mM MgCl₂ and 100 µM MnCl₂ as metal cofactors; when indicated, RPA was added to the reaction at a concentration of 100 or 500 nM. (b) PrimPol-Polε combined assay was carried out essentially as described in part (a) but using 50 µM MnCl₂, and when indicated adding Polε (10 nM) and RPA (20, 100 or 500 nM). After native agarose electrophoresis, the nature of the labelled products (circular ssDNA and linear ssDNA) was inferred from EtBr-staining of the gels prior to autoradiography. The schemes represent the stimulation of PrimPol primase and polymerase activities in the presence of RPA, and the subsequent stimulation on extension by Polε.