PROLIFERATING CELL NUCLEAR ANTIGEN PROMOTES MISINCORPORATION CATALYZED BY CALF THYMUS DNA-POLYMERASE-DELTA

A proliferating cell nuclear antigen (PCNA)-dependent complex, detecta ble after nondenaturing polyacrylamide gel electrophoresis, is formed between calf thymus DNA polymerase delta (pol delta) and synthetic oli gonucleotide template-primers containing a mispaired nucleotide at the 3'-terminal position of the primer, This complex is indistinguishable in composition from that formed with a fully base paired template-pri mer. Extension of a mispaired primer terminus is a component of DNA po lymerase fidelity, The fidelity of pol delta on synthetic oligonucleot ide template-primers was compared with and without its specific proces sivity factor, PCNA In the absence of PCNA, pol delta misincorporates less than one nucleotide for every 100,000 nucleotides incorporated co rrectly. Addition of PCNA to reactions reduces fidelity by at least 27 -fold, PCNA also confers upon pol delta, the ability to incorporate (a nd/or not excise) the dTTP analog, 2'-deoxythymidine-5'-O-(alpha-phosp honomethyl)- beta,gamma-diphosphate. A model is proposed whereby the i ncreased stability (decreased off-rate) of the pol delta . template-pr imer complex in the presence of PCNA facilitates unfavorable events ca talyzed by pol delta, This model suggests an explicit mechanistic requ irement for the intrinsic 3'-5'-exonuclease of pol delta.