Pif1 helicase directs eukaryotic Okazaki fragments toward the two-nuclease cleavage pathway for primer removal

Abstract

Eukaryotic Okazaki fragment maturation requires complete removal of the initiating RNA primer before ligation occurs. Polymerase delta (Pol delta) extends the upstream Okazaki fragment and displaces the 5'-end of the downstream primer into a single nucleotide flap, which is removed by FEN1 nuclease cleavage. This process is repeated until all RNA is removed. However, a small fraction of flaps escapes cleavage and grows long enough to be coated with RPA and requires the consecutive action of the Dna2 and FEN1 nucleases for processing. Here we tested whether RPA inhibits FEN1 cleavage of long flaps as proposed. Surprisingly, we determined that RPA binding to long flaps made dynamically by polymerase delta only slightly inhibited FEN1 cleavage, apparently obviating the need for Dna2. Therefore, we asked whether other relevant proteins promote long flap cleavage via the Dna2 pathway. The Pif1 helicase, implicated in Okazaki maturation from genetic studies, improved flap displacement and increased RPA inhibition of long flap cleavage by FEN1. These results suggest that Pif1 accelerates long flap growth, allowing RPA to bind before FEN1 can act, thereby inhibiting FEN1 cleavage. Therefore, Pif1 directs long flaps toward the two-nuclease pathway, requiring Dna2 cleavage for primer removal.

FIGURE 1.

Cleavage product distribution indicates both short and long products. A, initial FEN1 cleavage was assayed on the standard-44 substrate (U₂:T₄:D₁) in the presence of PCNA/RFC and either Pol δ-wt, denoted wt (lane 3), or Pol δ-01, denoted 01 (lane 4), as described under “Experimental Procedures.” The substrate is depicted above the figure. The asterisk indicates location of radiolabel. B, the size of flap cleavage products versus the intensity of the product band was graphed and moving average trendline added, as described under “Experimental Procedures.” The black line corresponds to the cleavage products for reconstitutions with Pol δ-wt. The gray line corresponds to the products for reconstitutions with Pol δ-01.

FIGURE 2.

FEN1 cleavage during flap displacement is moderately inhibited by RPA. Initial cleavage by FEN1 was assayed on the standard-44 substrate (U₂:T₄:D₁) in the presence of PCNA/RFC, Pol δ-01, and increasing amounts of RPA (50, 100, or 200 fmol) as described under “Experimental Procedures” (lanes 4–6). The substrate is depicted above the figure. Asterisk indicates location of radiolabel.

FIGURE 3.

RPA inhibits FEN1 cleavage of fixed flap substrates. A, cleavage by FEN1 was assayed in the presence of increasing amounts of RPA (50, 100, or 200 fmol) as described under “Experimental Procedures.” The 19-nt fixed double-flap (U₁:T₃:D₁)(lanes 1–5), 28-nt fixed double-flap (U₁:T₂:D₁)(lanes 6–10), and 37-nt fixed double-flap (U₁:T₁:D₁)(lanes 11–15) are depicted above the figure. The asterisk indicates location of radiolabel. B, the amount of RPA versus the percent cleavage inhibition was graphed for the 19-nt fixed double-flap (black boxes), 28-nt fixed double-flap (gray boxes), and 37-nt fixed double-flap (white boxes) for the gel in A. Percentages are indicated above the bar for each substrate at each concentration of RPA. C, cleavage by FEN1 was assayed in the presence of increasing amounts of RPA (50, 100, or 200 fmol) using the 37-nt fixed double-flap, as in A. In lanes 2–5, RPA was added at the start of the reaction and FEN1 was added after 5 min. In lanes 6–9, both FEN1 and RPA were added at the start of the reaction, as in lanes 12–15 of A. In lanes 10–13, FEN1 was added at the start of the reaction in the absence of Mg⁺², followed by addition of both RPA and Mg⁺² after 5 min. Lane 14 contains FEN1 alone in the absence of Mg⁺². The percent cleavage inhibition is shown under each lane.

FIGURE 4.

Pif1 improves flap displacement and promotes RPA inhibition of FEN1 cleavage. Initial cleavage by FEN1 was assayed on the standard-44 substrate (U₂:T₄:D₁) in the presence of PCNA/RFC, Pol δ-wt, and increasing amounts of Pif1 (25, 50, or 100 fmol) as described under “Experimental Procedures” (lanes 4–6). Cleavage was also assayed in the presence of RPA (100 fmol) and increasing amounts of Pif1 (25, 50, or 100 fmol) as described under “Experimental Procedures” (lanes 8–10). The substrate is depicted above the figure. Asterisk indicates location of radiolabel.

FIGURE 5.

Pif1-stimulated products are a small subset of cleavage products. The extent of FEN1 cleavage into the downstream primer was assayed on the standard-44 substrate (U₂:T₄:D₁) in the presence of PCNA/RFC, Pol δ-wt, and increasing amounts of Pif1 (25, 50, or 100 fmol) as described under “Experimental Procedures” (lanes 4–6). Cleavage was also assayed in the presence of RPA (100 fmol) and increasing amounts of Pif1 (25, 50, or 100 fmol) as described under “Experimental Procedures” (lanes 8–10). The substrate is depicted above the figure. Asterisk indicates location of radiolabel.

FIGURE 6.

RNA does not inhibit lengthening of flaps by Pif1 during synthesis of Okazaki fragments. A, initial cleavage by FEN1 was assayed on the RNA substrate (U₂:T₄:D₂) in the presence of PCNA/RFC, Pol δ-wt, and increasing amounts of Pif1 (25, 50, 100, or 200 fmol) as described under “Experimental Procedures” (lanes 5–8). Cleavage was also assayed in the presence of RPA (100 fmol) and increasing amounts of Pif1 (25, 50, 100, or 200 fmol) as described under “Experimental Procedures” (lanes 10–13). The substrate is depicted above the figure. The gray segment represents the ribonucleotide. The asterisk indicates location of radiolabel. B, the same reactions as in A except the substrate used was the methylated substrate (U₂:T₄:D₃). The substrate is depicted above the figure. The gray circle represents the 2′-O-methylated ribonucleotide. The asterisk indicates the location of radiolabel.

FIGURE 7.

Pif1 directs long flaps toward the two-nuclease pathway for primer removal. A, in most cases FEN1 is tracking near the base of the elongating flap, racing the displacement process to eventually reach the site of cleavage. Most RPA molecules would then bind behind the FEN1, too late to prevent cleavage (refer to Fig. 2, lanes 3–6). B, Pif1 promotes flap displacement, allowing some flaps to become long before FEN1 cleavage occurs (refer to Fig. 4, lanes 3–6). C, Pif1 increases the rate of flap formation such that FEN1 molecules take longer to track to the flap base for cleavage. In the presence of RPA, the flap can bind RPA ahead of the tracking FEN1. RPA bound in this position can prevent FEN1 cleavage (refer to Fig. 4, lanes 7–10).