Dynamics of enzymatic interactions during short flap human Okazaki fragment processing by two forms of human DNA polymerase δ

Lagging strand DNA replication requires the concerted actions of DNA polymerase delta, Fen1 and DNA ligase I for the removal of the RNA/DNA primers before ligation of Okazaki fragments. To better understand this process in human cells, we have reconstituted Okazaki fragment processing by the short flap pathway in vitro with purified human proteins and oligonucleotide substrates. We systematically characterized the key events in Okazaki fragment processing: the strand displacement, Pol delta/Fen1 combined reactions for removal of the RNA/DNA primer, and the complete reaction with DNA ligase I. Two forms of human DNA polymerase delta were studied: Pol delta 4 and Pol delta 3, which represent the heterotetramer and the heterotrimer lacking the p12 subunit, respectively. Pol delta 3 exhibits very limited strand displacement activity in contrast to Pol delta 4, and stalls on encounter with a 5'-blocking oligonucleotide. Pol delta 4 and Pol delta 3 exhibit different characteristics in the Pol delta/Fen1 reactions. While Pol delta 3 produces predominantly 1 and 2 nt cleavage products irrespective of Fen1 concentrations, Pol delta 4 produces cleavage fragments of 1-10 nts at low Fen1 concentrations. Pol delta 3 and Pol delta 4 exhibit comparable formation of ligated products in the complete system. While both are capable of Okazaki fragment processing in vitro, Pol delta 3 exhibits ideal characteristics for a role in Okazaki fragment processing. Pol delta 3 readily idles and in combination with Fen1 produces primarily 1 nt cleavage products, so that nick translation predominates in the removal of the blocking strand, avoiding the production of longer flaps that require additional processing. These studies represent the first analysis of the two forms of human Pol delta in Okazaki fragment processing. The findings provide evidence for the novel concept that Pol delta 3 has a role in lagging strand synthesis, and that both forms of Pol delta may participate in DNA replication in higher eukaryotic cells. (C) 2013 Elsevier B.V. All rights reserved.