Flexibility of Eukaryotic Okazaki Fragment Maturation through Regulated Strand Displacement Synthesis

Okazaki fragment maturation to produce continuous lagging strands in eukaryotic cells requires precise coordination of strand displacement synthesis by DNA polymerase ; (Pol ;) with 5·-flap cutting by FEN1RAD27 endonuclease. Excessive strand displacement is normally prevented by the 3·-exonuclease activity of Pol ;. This core maturation machinery can be assisted by Dna2 nuclease/helicase that processes long flaps. Our genetic studies show that deletion of the POL32 (third subunit of Pol ;) or PIF1 helicase genes can suppress lethality or growth defects of rad27Δ pol3-D520V mutants (defective for FEN1RAD27 and the 3·-exonuclease of Pol ;) that produce long flaps and of dna2Δ mutants that are defective in cutting long flaps. On the contrary, pol32Δ or pif1Δ caused lethality of rad27Δ exo1Δ double mutants, suggesting that Pol32 and Pif1 are required to generate longer flaps that can be processed by Dna2 in the absence of the short flap processing activities of FEN1RAD27 and Exo1. The genetic analysis reveals a remarkable flexibility of the Okazaki maturation machinery and is in accord with our biochemical analysis. In vitro, the generation of short flaps by Pol ; is not affected by the presence of Pol32; however, longer flaps only accumulate when Pol32 is present. The presence of FEN1RAD27 during strand displacement synthesis curtails displacement in favor of flap cutting, thus suggesting an active hand-off mechanism from Pol ; to FEN1RAD27. Finally, RNA-DNA hybrids are more readily displaced by Pol ; than DNA hybrids, thereby favoring degradation of initiator RNA during Okazaki maturation. Okazaki fragment maturation to produce continuous lagging strands in eukaryotic cells requires precise coordination of strand displacement synthesis by DNA polymerase ; (Pol ;) with 5·-flap cutting by FEN1RAD27 endonuclease. Excessive strand displacement is normally prevented by the 3·-exonuclease activity of Pol ;. This core maturation machinery can be assisted by Dna2 nuclease/helicase that processes long flaps. Our genetic studies show that deletion of the POL32 (third subunit of Pol ;) or PIF1 helicase genes can suppress lethality or growth defects of rad27Δ pol3-D520V mutants (defective for FEN1RAD27 and the 3·-exonuclease of Pol ;) that produce long flaps and of dna2Δ mutants that are defective in cutting long flaps. On the contrary, pol32Δ or pif1Δ caused lethality of rad27Δ exo1Δ double mutants, suggesting that Pol32 and Pif1 are required to generate longer flaps that can be processed by Dna2 in the absence of the short flap processing activities of FEN1RAD27 and Exo1. The genetic analysis reveals a remarkable flexibility of the Okazaki maturation machinery and is in accord with our biochemical analysis. In vitro, the generation of short flaps by Pol ; is not affected by the presence of Pol32; however, longer flaps only accumulate when Pol32 is present. The presence of FEN1RAD27 during strand displacement synthesis curtails displacement in favor of flap cutting, thus suggesting an active hand-off mechanism from Pol ; to FEN1RAD27. Finally, RNA-DNA hybrids are more readily displaced by Pol ; than DNA hybrids, thereby favoring degradation of initiator RNA during Okazaki maturation. The process of DNA replication in eukaryotic cells leads to the generation of a vast number of Okazaki fragments on the lagging strand of the replication fork. Approximately 50,000,000 Okazaki fragments are synthesized when a human cell replicates, and all of these need to be efficiently and accurately matured into continuous lagging strands to ensure genome integrity. Various DNA structures are generated during the synthesis and maturation of Okazaki fragments. These structures constitute the largest pool for potential DNA damage in the cell. Incomplete or poorly processed Okazaki fragments can lead to repeat expansion mutations, small duplication mutations, and to the generation of double-stranded DNA breaks (1Gordenin D.A. Kunkel T.A. Resnick M.A. Nat. Genet. 1997; 16: 116-118Crossref PubMed Scopus (189) Google Scholar). A large number of activities have been implicated in lagging strand DNA maturation. In the budding yeast Saccharomyces cerevisiae, the RAD27 gene product is the 5′-flap endonuclease FEN1. FEN1RAD27 has been assigned a dominant role in creating ligatable nicks during Okazaki maturation (reviewed in Refs. 2Liu Y. Kao H.I. Bambara R.A. Annu. Rev. Biochem. PubMed Scopus Google and PubMed Scopus Google Scholar). for the of RAD27 in Okazaki maturation by the of small to Pol DNA polymerase Pol Pol ; with Pol and Pol Pol32; polymerase of Pol of Pol replication replication cell in by short in mutants 1997; PubMed Scopus Google Scholar). 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