Fig. 4: Impact of RPA binding on PolD and PriSL primer extension activity.

A 17 nucleotide-long primer labeled with a 5′ Cy5 fluorophore annealed to a 87 nucleotide-long template was used as substrate in reactions with all deoxyribonucleotides and ribonucleotides at physiological concentrations (sequences are shown in Supplementary Table 4). Reactions were incubated for 10 min at 55 °C with PolD or PriSL, and one of several RPA constructs. Experiments were repeated n = 4 times, band integration was performed in all 4 biological replicates (87 nt band for PolD gels, >70 nt bands for PriSL gels) to derive standard deviation. Each bar shows the mean value, standard deviation is represented as error bars, and individual measurements are shown as white dots. Uncropped gels are provided as a Source Data file. a–d Impact of RPA binding on PolD primer-extension activity. PolD was incubated at a concentration of 0.25 µM, with increasing amounts of different RPA constructs ranging from 0.05 to 0.8 µM (lanes 4-8). Lanes 1 and 2 contain an oligonucleotide ladder of 87-nt and a negative control experiment without proteins, respectively. Lane 3 contains PolD (0.25 µM) in the absence of RPA. a primer extension assay by PolD in the presence of RPA, b of Rpa2^WH, c of RPA^ΔWH and d RPA^ΔWH + Rpa2^WH. e Impact of RPA binding on PriSL primer-extension activity. The primer-template substrate was the same as in (a). PriSL (0.2 µM) was incubated with increasing amounts of different RPA constructs ranging from 0.05 to 0.8 µM (lanes 3–7). Lane 1 is the negative control without proteins. Lane 2 contains PriSL (0.2 µM) without RPA. Lane 8 contains oligonucleotide ladders (87 nt and 57-nt). From the left to the right panels: e primer extension assay by PriSL in the presence of RPA, f of Rpa2^WH, g of RPA^ΔWH and h RPA^ΔWH + Rpa2^WH. The short ~20 nt PriSL primer extension products (black arrowheads) and the PolD exonuclease digestion products (*) are highlighted.