Impairment of proliferating cell nuclear antigen-dependent apurinic/apyrimidinic site repair on linear DNA

Repair of apurinic/apyrimidinic (AP) sites by mammalian cell extracts was compared using circular and linear DNA substrates. Extracts prepared from DNA polymerase beta (pol beta)-proficient mouse fibroblasts repaired AP sites on both circular and linear DNA. However, extracts from the isogenic pol beta-knockout cells repaired AP sites on circular DNA but not efficiently on linear DNA. The circularity-dependent repair by the pol beta-knockout cell extract was completely inhibited by antiproliferating cell nuclear antigen (PCNA) antibody but fully restored by addition of purified PCNA. Pretreatment of the linear DNA with AP endonuclease did not improve repair, indicating that impairment of AP site repair on linear DNA by pol beta-knockout cell extracts is not due to inefficiency of damage incision but rather to deficiency at the subsequent steps. These results indicate that AP sites can be repaired on circular DNA by the PCNA-dependent pathway in addition to the pol beta-dependent pathway and that the PCNA-dependent repair mechanism is poorly functional on linear DNA in vitro.