Okazaki fragment processing: modulation of the strand displacement activity of DNA polymerase delta by the concerted action of replication protein A, proliferating cell nuclear antigen, and flap endonuclease-1

Abstract

DNA polymerase (pol) delta is essential for both leading and lagging strand DNA synthesis during chromosomal replication in eukaryotes. Pol delta has been implicated in the Okazaki fragment maturation process for the extension of the newly synthesized fragment and for the displacement of the RNA/DNA segment of the preexisting downstream fragment generating an intermediate flap structure that is the target for the Dna2 and flap endonuclease-1 (Fen 1) endonucleases. Using a single-stranded minicircular template with an annealed RNA/DNA primer, we could measure strand displacement by pol delta coupled to DNA synthesis. Our results suggested that pol delta alone can displace up to 72 nucleotides while synthesizing through a double-stranded DNA region in a distributive manner. Proliferating cell nuclear antigen (PCNA) reduced the template dissociation rate of pol delta, thus increasing the processivity of both synthesis and strand displacement, whereas replication protein A (RP-A) limited the size of the displaced fragment down to 20-30 nucleotides, by generating a "locked" flap DNA structure, which was a substrate for processing of the displaced fragment by Fen 1 into a ligatable product. Our data support a model for Okazaki fragment processing where the strand displacement activity of DNA polymerase delta is modulated by the concerted action of PCNA, RP-A and Fen 1.

Figure 1

Minicircular template-primer to study strand displacement-coupled DNA synthesis by pol δ. See text for details.

Figure 2

Pol δ shows strand displacement activity on the 72:17-mer minicircular template-primer. (A) Pol δ (0.1 unit), was incubated in the presence of 12 nM (3′-OH ends) 72:17-mer template-primer under the conditions described in Materials and Methods. Reactions were stopped at the indicated times and the products resolved on a 14% PAA/7 M Urea gel. Lane M: 5′-labeled linear 70-mer and 17-mer were loaded as markers. (B) Sizes of the synthesized products in nt were calculated from their relative electrophoretic mobility and plotted versus time. Data points were fitted to a mixed exponential equation by computer simulation. (C) Length (in nt) of the displaced strand was calculated from the size of the synthesized products and plotted versus time. The solid line represents the best linear interpolation of the data points.

Figure 3

RP-A limits the size of the DNA strand displaced by pol δ. (A) Pol δ (0.1 unit), was incubated in the presence of 12 nM (3′-OH ends) 72:17-mer template-primer and 16 nM RP-A (as heterotrimer), under the conditions described in Materials and Methods. Reactions were stopped at the indicated time points and the products were resolved on a 14% PAA/7 M Urea gel. Lane M: 5′-labeled linear 70-mer and 17-mer were loaded as markers. (B) Pol δ (0.1 unit) was incubated in the presence of 12 nM (3′-OH ends) 72:17-mer template-primer and increasing amounts of RP-A under the conditions described in Materials and Methods. Parallel reactions were stopped after 40 s (lanes 1–4) or 340 s (lanes 5–7) and the products were resolved on a 14% PAA/7 M Urea gel. Lane M: 5′-labeled linear 70-mer and 17-mer were loaded as markers. (C) Pol δ (0.1 unit) was incubated in the presence of 12 nM (3′-OH ends) 72:17-mer template-primer under the conditions described in Materials and Methods. Sixteen nanomoles of nM RP-A were added either at the beginning of the reaction or at the indicated times. Reactions were stopped after 5 min and the products were resolved on a 14% PAA/7 M Urea gel. Densitometric analysis of the bands corresponding to the products longer than 90 nt allowed the calculation of their relative amounts. Band intensities (I) were corrected for background and normalized to the total through the equation: relative I = (I_n/(I_n-1 + . . . + I_n-i))/Σ(I_i)_{i = 1… n}, where I_n is the intensity at the position of interest (n), (I_n-1 + . . . + I_n-i) is the sum of the intensities of all bands at positions below n, and Σ(I_i)_{i = 1… n} is the sum of the intensities of all of the bands in the lane. These values were then plotted in dependence of the times of addition of RP-A.

Figure 4

PCNA increases the processivity of strand displacement-coupled DNA synthesis by pol δ. (A) Pol δ (0.1 unit) was incubated in the presence of 12 nM (3′-OH ends) 72–5′-³²P-labeled 17-mer template-primer under the conditions described in Materials and Methods for single turnover synthesis. Reactions were stopped at the indicated time points and the products were resolved on a 14% PAA/7 M Urea gel. Linear 5′-labeled 70- and 17-mer markers migrated at the positions indicated by arrows. (B) Pol δ (0.1 unit) was incubated in the presence of 12 nM (3′-OH ends) 72:17-mer template-primer in the absence or presence of RP-A (16 nM), RF-C (0.02 unit), and PCNA (1 μM as trimer), under the conditions described in Materials and Methods. Reactions were stopped at the indicated times and the products were resolved on a 14% PAA/7 M Urea gel. Arrows indicate the position of 85- and 17-mer markers, as well as the length of the different products synthesized.

Figure 5

Fen 1 can process the ssDNA flap generated by pol δ strand displacement activity into ligatable products. (A) Dose-dependent processing of the ssDNA displaced flap by Fen 1. Pol δ (0.1 unit) was incubated in the presence of 12 nM (3′-OH ends) 72:17-mer template-primer, 16 nM RP-A (as heterotrimer), RF-C (0.02 units), PCNA (1 μM as trimer), and in the absence (lane 1) or presence of 0.2 μM (lane 2) or 1 μM (lane 3) of Fen 1, under the conditions described in Materials and Methods. Reactions were stopped after a 10-min incubation and the products were resolved on a 14% PAA/7 M Urea gel. Sizes of the DNA products are indicated at the left. (B) Ability of Fen 1 wild type and mutants to process the ssDNA displaced flap. Pol δ (0.1 unit) was incubated in the presence of 12 nM (3′-OH ends) 72:17-mer template-primer, 16 nM RP-A (as heterotrimer), RF-C (0.02 units), PCNA (1 μM as trimer), and in the absence (lane 1) or presence of 1 μM of Fen 1 wild type (lane 2), Fen 1 ΔC (lane 3), or Fen 1 D86A (lane 4), under the conditions described in Materials and Methods. Reactions were stopped after a 10-min incubation and the products were resolved on a 14% PAA/7 M Urea gel. Size of the DNA products are indicated at the left. (C) Fen 1 generates ligatable products. Pol δ (0.1 unit) was incubated in the presence of 12 nM (3′-OH ends) 72:27-mer template-RNA/DNA hybrid primer, 25 nM RP-A (as heterotrimer), RF-C (0.1 unit), and PCNA (0.2 μM as trimer) and in the absence (lanes 3 and 4) or presence (lanes 1 and 2) of 100 nM sFen 1, under the conditions described in Materials and Methods. Reactions were stopped after a 10-min incubation and the products were resolved on a 14% PAA/7 M Urea gel. Size of the DNA products are indicated at the left. Lane 1: after a 10-min incubation, sample was heat-inactivated for 20 min at 75°C, cooled down at 37°C, and then T4DNA ligase was added along with ligase reaction buffer. After a 30-min incubation at 37°C, sample was stopped and processed as above. ssl, single-stranded linear DNA; ssc, single-stranded circular DNA.