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. 2001 Nov 27;98(25):14298–14303. doi: 10.1073/pnas.251193198

Figure 5.

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.

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