Proliferating cell nuclear antigen promotes misincorporation catalyzed by calf thymus DNA polymerase delta.

Abstract:

A proliferating cell nuclear antigen (PCNA)-dependent complex, detectable after nondenaturing polyacrylamide gel electrophoresis, is formed between calf thymus DNA polymerase delta (pol delta) and synthetic oligonucleotide 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-primer. Extension of a mispaired primer terminus is a component of DNA polymerase fidelity. The fidelity of pol delta on synthetic oligonucleotide template-primers was compared with and without its specific processivity factor, PCNA. In the absence of PCNA, pol delta misincorporates less than one nucleotide for every 100,000 nucleotides incorporated correctly. Addition of PCNA to reactions reduces fidelity by at least 27-fold. PCNA also confers upon pol delta, the ability to incorporate (and/or not excise) the dTTP analog, 2'-deoxythymidine-5'-O-(alpha-phosphonomethyl)-beta, gamma-diphosphate. A model is proposed whereby the increased stability (decreased off-rate) of the pol delta.template-primer complex in the presence of PCNA facilitates unfavorable events catalyzed by pol delta. This model suggests an explicit mechanistic requirement for the intrinsic 3'-5'-exonuclease of pol delta.

Polymerases:

Topics:

Nucleotide Incorporation

Status:

new topics/pols set partial results complete validated

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