PCNA is efficiently loaded on the DNA recombination intermediate to modulate polymerase δ, η, and ζ activities

Abstract

Proliferating cell nuclear antigen (PCNA) is required for DNA homologous recombination (HR), but its exact role is unclear. Here, we investigated the loading of PCNA onto a synthetic D-loop (DL) intermediate of HR and the functional interactions of PCNA with Rad51 recombinase and DNA polymerase (Pol) δ, Pol η, and Pol ζ. PCNA was loaded onto the synthetic DL as efficiently as it was loaded onto a primed DNA substrate. Efficient PCNA loading requires Replication Protein A, which is associated with the displaced ssDNA loop and provides a binding site for the clamp-loader Replication Factor C. Loaded PCNA greatly stimulates DNA synthesis by Pol δ within the DL but does not affect primer recognition by Pol δ. This suggests that the essential role of PCNA in HR is not recruitment of Pol δ to the DL but stimulation of Pol δ to displace a DNA strand during DL extension. Both Pol η and Pol ζ extended the DL more efficiently than Pol δ in the absence of PCNA, but little or no stimulation was observed in the presence of PCNA. Finally, Rad51 inhibited both the loading of PCNA onto the DL and the extension of the DL by Pol δ and Pol η. However, preloaded PCNA on the DL counteracts the Rad51-mediated inhibition of the DL extension. This suggests that the inhibition of postinvasion DNA synthesis by Rad51 occurs mostly at the step of PCNA loading.