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

Abstract

Lagging strand DNA replication requires the concerted actions of DNA polymerase δ, 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 δ/Fen1 combined reactions for removal of the RNA/DNA primer, and the complete reaction with DNA ligase I. Two forms of human DNA polymerase δ were studied: Pol δ4 and Pol δ3, which represent the heterotetramer and the heterotrimer lacking the p12 subunit, respectively. Pol δ3 exhibits very limited strand displacement activity in contrast to Pol δ4, and stalls on encounter with a 5’-blocking oligonucleotide. Pol δ4 and Pol δ3 exhibit different characteristics in the Pol δ/Fen1 reactions. While Pol δ3 produces predominantly 1 and 2 nt cleavage products irrespective of Fen1 concentrations, Pol δ4 produces cleavage fragments of 1–10 nts at low Fen1 concentrations. Pol δ3 and Pol δ4 exhibit comparable formation of ligated products in the complete system. While both are capable of Okazaki fragment processing in vitro, Pol δ3 exhibits ideal characteristics for a role in Okazaki fragment processing. Pol δ3 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 δ in Okazaki fragment processing. The findings provide evidence for the novel concept that Pol δ3 has a role in lagging strand synthesis, and that both forms of Pol δ may participate in DNA replication in higher eukaryotic cells.

The tail that wags the dog: p12, the smallest subunit of DNA polymerase δ, is degraded by ubiquitin ligases in response to DNA damage and during cell cycle progression

“…We completed a contemporaneous analysis of the reactions involved in Okazaki fragment processing by both forms of Pol δ in cooperation with Fen1. 22 This study revealed that Pol δ3 possesses characteristics that are ideal for Okazaki fragment processing, further supporting the idea that it participates in DNA replication as well as in DNA repair.…”

“…3, panel II). Our study of the combined actions of Pol δ3 and Fen1 shows that this is predominantly a nick translation process, while that of Pol δ4 and Fen1 presents as either a short flap pathway or a nick translation process that is dependent on the Fen1 concentration 22 (Fig. 3).…”

“…this diagram summarizes the reactions of Pol δ4 and Pol δ3 with Fen1 in the reconstituted system for Okazaki fragment processing. 22 Panel i (blue) shows the operation of the short flap pathway. in reaction "1", Pol δ extends the primer until it meets a 5′ downstream primer.…”

“…22 The processing of Okazaki fragments is essentially one of primer removal and replacement by Pol δ and Fen1. [75][76][77][78] We used rigorously purified human enzymes and model substrates to compare the behavior of Pol δ4 and Pol δ3 in a reconstituted system for Okazaki fragment processing.…”

Regulation and Modulation of Human DNA Polymerase δ Activity and Function

“…These include an increase in processivity, possibly because PDIP46 may stabilize Pol δ binding to PCNA by a bridging interaction, as well as by a direct activation that involves alteration of the kinetic properties of Pol δ4. The effects of PDIP46 were examined on model oligonucleotide templates [127] in assays that examined primer extension and strand displacement in order to gain insights into its mechanism(s) of action [56]. In the absence of PCNA, Pol δ4 behaves in a distributive fashion, and PDIP46 clearly stimulates this activity.…”