This pathway involves limited strand displacement by Pol δ, which creates short flaps (1–8 nts) that are cleavable by flap endonuclease 1 (Fen1). The major pathway for the removal of the RNA/DNA primer is the short flap pathway. Thus, both Fen1 and Pol δ are recruited to sites of nucleotide excision repair with similar kinetics in in vitro assays, while Fen1 also plays a role in base excision and mismatch repair. In addition, the processes of gap filling in DNA repair processes such as during nucleotide excision repair also bears resemblances to Pol δ/Fen1 reactions. Thus, the removal of these ends during Okazaki fragment processing can be considered in the broader perspective of a replication-coupled DNA repair process. The 5’ ends of Okazaki fragments include DNA synthesized by Pol α (which is less accurate than Pol δ as it lacks a proof reading 3’ to 5’ exonuclease) which are likely to contain errors. Thus, the process requires the removal of the RNA/DNA primer when it is encountered by a new Okazaki fragment and the formation of a nick (reviewed in ) that can be sealed by DNA ligase I ). These primers must be removed before ligation of the Okazaki fragment to the lagging strand, because of their RNA content and also because Pol α does not possess a proofreading 3’ to 5’ exonuclease and is error prone. The synthesis of eukaryotic Okazaki fragments starts with the generation of RNA/DNA primers (8–12 RNA nts followed by 10–20 DNA nts) by Pol α/primase, which are extended by the replicative proofreading DNA polymerases. In eukaryotes, the Okazaki fragments are only about 200 nt in length. The length of Okazaki fragments differs from prokaryotes to eukaryotes. Because of the different polarity of the DNA strands, the leading strand is continuously extended in the 5’ to 3’ direction, while the lagging strand is discontinuously synthesized by the production and joining of Okazaki fragments. The process of DNA replication at the replication fork requires that the two daughter strands be coordinately synthesized. 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. These studies represent the first analysis of the two forms of human Pol δ 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. While both are capable of Okazaki fragment processing in vitro, Pol δ3 exhibits ideal characteristics for a role in Okazaki fragment processing.
Pol δ3 and Pol δ4 exhibit comparable formation of ligated products in the complete system. 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 δ4 and Pol δ3 exhibit different characteristics in the Pol δ/Fen1 reactions. Pol δ3 exhibits very limited strand displacement activity in contrast to Pol δ4, and stalls on encounter with a 5’-blocking oligonucleotide. 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. 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. 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. 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.
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