Figure 6. RPA recruits PrimPol to stalled replication forks in vivo.

(a) PrimPol's RBM-A, but not RBM-B, is critical for recruitment to chromatin. Flp-In T-Rex-293 cells transfected with WT and RBM-A and B mutant PrimPol constructs were either mock (−) or ultraviolet-C (30 J m⁻²) (+) irradiated before separation into Triton X-100 (0.5%) soluble and insoluble fractions. Samples were analysed by western blot alongside whole-cell extracts. Only insoluble samples are presented here, whole-cell extracts and soluble blots can be found in Supplementary Fig. 6. (b) PrimPol's RBD is recruited to Xenopus egg extract chromatin in response to aphidicolin treatment, RBM-A is critical for this recruitment. Recombinant hPrimPol GST constructs (4 ng μl⁻¹) were added to Xenopus egg extract supplemented with sperm nuclei (3 × 10³ μl⁻¹). Extract was treated with aphidicolin 100 μg ml⁻¹ and incubated at 21 °C for 80 min. Chromatin was isolated and associated proteins analysed by SDS–PAGE and western blotting using the antibodies indicated. (c) Low concentrations of RPA stimulate PrimPol's primase activity, high concentrations inhibit. Primer synthesis by WT hPrimPol (400 nM) on M13 ssDNA templates (20 ng μl⁻¹) in the presence of increasing concentrations of RPA. ‘C' indicates the no enzyme control, oligonucleotide (Nt) length markers are shown on the left of the gel. (d) Quantification of data shown in ‘c'. For each RPA concentration the fold increase in primer synthesis relative to reactions containing no RPA was calculated. Data are representative of three repeat experiments. (e) Schematic showing the effect of increasing RPA concentrations on PrimPol's primase activity. When no RPA is present a proportion of PrimPol binds to the M13 template and facilitates primer synthesis (left). When low/moderate concentrations of RPA are present PrimPol is recruited to the RPA/ssDNA interface causing an increase in primer synthesis activity (middle). At high RPA concentrations the M13 DNA template is fully saturated, blocking access of PrimPol to the DNA and inhibiting primer synthesis (right).