Evidence for the hypothesis that two forms of human DNA polymerase δ participate in DNA replication (925.2)

Abstract

Eukaryotic DNA polymerase δ (Pol δ) is the polymerase primarily involved in lagging strand DNA synthesis. Human Pol δ is a heterotetramer (Pol δ4) composed of four subunits. The p12 subunit is degraded in response to DNA damage, resulting in the in vivo conversion of Pol δ4 to Pol δ3, the trimer lacking p12. Thus, Pol δ3 is the form that is involved in DNA repair. The hypothesis that Pol δ3 is also capable of performing lagging strand DNA synthesis was tested in a reconstituted system for Okazaki fragment processing using purified enzymes and model oligonucleotide substrates. In the combined Pol δ3/Fen1 reactions, the products formed by removal of the downstream blocking primer were almost exclusively mononucleotides, showing that the reaction proceeded by nick translation, and was not affected by Fen1 concentrations. With Pol δ4/Fen1, the products were 1‐10 nts at low Fen1 concentrations, showing that the reaction proceeded by the short flap pathway. As Fen1 concentrations were increased the product size was shifted to mono‐nucleotides, i.e.,. to a nick translation mode. These studies show that Pol δ3 is ideally suited for Okazaki fragment processing. In parallel studies, we have shown that p12 is degraded during the G1/S transition so that a mixture of Pol δ4 and Pol δ3 is present in S phase, under the control of CRL4Cdt2, which also regulates the degradation of the licensing factors Cdt1, Set8 and p21.Grant Funding Source: Supported by GM31973 and ES14737