The p12 Subunit of Human Polymerase δ Modulates the Rate and Fidelity of DNA Synthesis

Abstract

This study examines the role of the p12 subunit in the function of the human DNA polymerase δ (Pol δ) holoenzyme by comparing the kinetics of DNA synthesis and degradation catalyzed by the four-subunit complex, the three-subunit complex lacking p12, and site-directed mutants of each lacking proofreading exonuclease activity. Results show that p12 modulates the rate and fidelity of DNA synthesis by Pol δ. All four complexes synthesize DNA in a rapid burst phase and a slower, more linear phase. In the presence of p12, the burst rates of DNA synthesis are ∼5 times faster, while the affinity of the enzyme for its DNA and dNTP substrates appears unchanged. The p12 subunit alters Pol δ fidelity by modulating the proofreading 3′ to 5′ exonuclease activity. In the absence of p12, Pol δ is more likely to proofread DNA synthesis because it cleaves single-stranded DNA twice as fast and transfers mismatched DNA from the polymerase to the exonuclease sites 9 times faster. Pol δ also extends mismatched primers 3 times more slowly in the absence of p12. Taken together, the changes that p12 exerts on Pol δ are ones that can modulate its fidelity of DNA synthesis. The loss of p12, which occurs in cells upon exposure to DNA-damaging agents, converts Pol δ to a form that has an increased capacity for proofreading.