The Saccharomyces cerevisiae Dna2 can function as a sole nuclease in the processing of Okazaki fragments in DNA replication

Abstract

During DNA replication, synthesis of the lagging strand occurs in stretches termed Okazaki fragments. Before adjacent fragments are ligated, any flaps resulting from the displacement of the 5' DNA end of the Okazaki fragment must be cleaved. Previously, Dna2 was implicated to function upstream of flap endonuclease 1 (Fen1 or Rad27) in the processing of long flaps bound by the replication protein A (RPA). Here we show that Dna2 efficiently cleaves long DNA flaps exactly at or directly adjacent to the base. A fraction of the flaps cleaved by Dna2 can be immediately ligated. When coupled with DNA replication, the flap processing activity of Dna2 leads to a nearly complete Okazaki fragment maturation at sub-nanomolar Dna2 concentrations. Our results indicate that a subsequent nucleolytic activity of Fen1 is not required in most cases. In contrast Dna2 is completely incapable to cleave short flaps. We show that also Dna2, like Fen1, interacts with proliferating cell nuclear antigen (PCNA). We propose a model where Dna2 alone is responsible for cleaving of RPA-bound long flaps, while Fen1 or exonuclease 1 (Exo1) cleave short flaps. Our results argue that Dna2 can function in a separate, rather than in a Fen1-dependent pathway.

Figure 1.

Dna2 cleaves DNA near a base of a flap. (A) Nuclease assay. *, radioactive label. (B) Wild-type (wt), nuclease-dead (E675A) or double-dead (E675A/K1080E, EA/KE) Dna2 variants (all 2 nM) were incubated with a DNA substrate containing a 30 nt-long flap, in the presence of RPA in a buffer containing 2 mM magnesium acetate. The reaction products were separated on 20% polyacrylamide denaturing urea gel. Cleavage at the base of the flap produces a fragment of 32 nt in length (position 0). (C) Increasing concentrations of Dna2 were incubated with a substrate containing a 30 nt-long flap as in (B), but in 10 mM magnesium acetate buffer, with or without RPA, as indicated. §, substrate cleaved by Dna2 in the absence of RPA. (D) Quantification of experiments such as in (C). Averages shown, n = 2; error bars, s.e.m. (E) Quantitation of products cleaved within 5 nt of flap base from (C). Averages shown, n = 2; error bars, s.e.m. (F) Experiment as in (C), but with helicase-dead Dna2 K1080E variant in the presence of RPA.

Figure 2.

The product of Dna2 can be directly ligated. (A) Assay. *, radioactive label. (B) Increasing concentrations of wild-type Dna2 were incubated with a substrate containing a 30 nt-long flap with or without Lig1, in the presence of RPA. The reaction products were separated on a 20% polyacrylamide denaturing urea gel. Cleavage at the base of the flap produces a fragment of 32 nt in length. Ligation of the cleaved intermediate results in a final product of 51 nt in length. (C) Quantitation of data such as in (B), with wild-type or mutants of Dna2 (nuclease-dead, E675A; helicase and nuclease-dead, EA/KE). The ³²P label was placed either at the 3′ terminus of the flapped oligonucleotide or at the 5′ terminus of the upstream primer. Averages shown, n = 2; error bars, s.e.m. (D) Quantitation of experiments such as in (B), but with helicase-dead Dna2 K1080E. The ³²P label was placed either at the 3′ terminus of the flapped oligonucleotide or at the 5′ terminus of the upstream primer. Averages shown, n = 2; error bars, s.e.m.

Figure 3.

Dna2 is highly efficient in flap processing during replication. (A) Replication assay. See ‘Materials and Methods’ section for details. (B) Dna2 is required for the completion of replication of a substrate containing a 5′ ssDNA flap of 30 nt in length. Reactions contained pol δ, PCNA, RFC, RPA, Lig1 and Dna2 as indicated. Positions of substrate ssDNA, open circular intermediate (ocDNA) and closed circular supercoiled final product (scDNA) are indicated. Final product appears in a Dna2 concentration dependent manner. (C) Quantitation of data such as in (B). Averages shown, n = 3; error bars, s.e.m. (D) Pol δ, RFC, PCNA and RPA are required for DNA synthesis, Dna2 and Lig1 for the formation of scDNA product. A ‘complete’ reaction contained all components as described in (B) without Dna2. Proteins were omitted from the reactions as indicated. (E) Nuclease activity of Dna2 is required for flap processing. Nuclease- and helicase-dead Dna2 E675A/K1080E variant was used where indicated. (F) Same assay as in (E), but using a substrate without a flap. Dna2 was not required for the processing of the flap-less substrate. A ‘complete’ reaction contained all components as described in (B) without Dna2. Marker, a sample containing DNA species corresponding to scDNA, linear DNA and ocDNA. (G) Replication assay with a flap-less substrate. Protein components were omitted from the reactions where indicated. A ‘complete’ reaction contained all components as described in (B) without Dna2.

Figure 4.

Effect of pol δ exonuclease activity on Okazaki fragment processing by Dna2 and Fen1. (A) Replication assay with a 30 nt-long flapped primer. (B) Reactions contained RFC, PCNA, RPA, Lig1 and Dna2, where indicated. Pol δ wild-type or the exonuclease-deficient mutant (pol δ exo−) were titrated into the reactions. Positions of substrate ssDNA, open circular intermediate (ocDNA) and closed circular supercoiled final product (scDNA) are indicated. In the presence of pol δ exo−, Dna2 stimulates the completion of replication only to a minor extent. (C) Quantitation of data such as in (B). Averages shown, n = 2; error bars, s.e.m.

Figure 5.

Effect of Fen1 on the flap processing by Dna2. (A) Assay. *, radioactive label. (B) Increasing concentrations of Fen1 were used to supplement reactions containing RPA, and Dna2 or Lig1 as indicated. The presence of Fen1 led to a decrease of the final ligated reaction product. (C) Quantitation of data such as in (B). Averages shown, n = 2; error bars, s.e.m. (D) Replication assay. (E) Kinetic replication reactions contained pol δ, PCNA, RFC, RPA, Lig1 and either 0.13 nM Dna2 (left part) or 0.13 nM Dna2 and 0.13 nM Fen1 (right part). Positions of substrate ssDNA, open circular intermediate (ocDNA) and closed circular supercoiled final product (scDNA) are indicated on the right. Samples were terminated at various time points, as indicated. (F) Same assay as in (E) but with 0.13 nM Dna2 and 1.3 nM Fen1. (G) Quantitation of experiments such as in (E and F). Averages shown, n = 3; error bars, s.e.m.

Figure 6.

Model of Okazaki fragment processing in eukaryotes. In cases when flaps formed upon the strand displacement activity of pol δ are long enough to bind RPA, Dna2 alone is primarily responsible for their processing in most cases (left). Short flaps are primarily processed by Fen1, Exo1 or possibly other nucleases (middle and right). See text for details.