DNA polymerase switching: I. Replication factor C displaces DNA polymerasealpha prior to PCNA loading

An important not yet fully understood event in DNA replication is the DNA p olymerase (pol) switch from pol alpha to pol delta. Indirect evidence sugge sted that the clamp loader replication factor C (RF-C) plays an important r ole, since a replication competent protein complex containing pol alpha, po l delta and RF-C could perform pol switching in the presence of proliferati ng cell nuclear antigen (PCNA). By using purified pol alpha/primase, pol de lta, RF-C, PCNA and RP-A we show that: (i) RF-C can inhibit pol alpha in th e presence of ATP prior to PCNA loading, (ii) RF-C decreases the affinity o f poi a for the 3'OH primer ends, (iii) the inhibition of pol alpha by RF-C is released upon PCNA loading, (iv) ATP hydrolysis is required for PCNA lo ading and subsequent release of inhibition of pol alpha, (v) under these co nditions a switching from pol alpha/primase to pol delta is evident. Thus, RF-C appears to be critical for the pol alpha to pol delta switching. Based on these results, a model is proposed in which RF-C induces the poi switch ing by sequestering the 3'-OH end from pol alpha and subsequently recruitin g PCNA to DNA. (C) 2000 Academic Press.