Acetylation of Dna2 endonuclease/helicase and flap endonuclease 1 by p300 promotes DNA stability by creating long flap intermediates

Abstract

Flap endonuclease 1 (FEN1) and Dna2 endonuclease/helicase (Dna2) sequentially coordinate their nuclease activities for efficient resolution of flap structures that are created during the maturation of Okazaki fragments and repair of DNA damage. Acetylation of FEN1 by p300 inhibits its endonuclease activity, impairing flap cleavage, a seemingly undesirable effect. We now show that p300 also acetylates Dna2, stimulating its 5'-3' endonuclease, the 5'-3' helicase, and DNA-dependent ATPase activities. Furthermore, acetylated Dna2 binds its DNA substrates with higher affinity. Differential regulation of the activities of the two endonucleases by p300 indicates a mechanism in which the acetylase promotes formation of longer flaps in the cell at the same time as ensuring correct processing. Intentional formation of longer flaps mediated by p300 in an active chromatin environment would increase the resynthesis patch size, providing increased opportunity for incorrect nucleotide removal during DNA replication and damaged nucleotide removal during DNA repair. For example, altering the ratio between short and long flap Okazaki fragment processing would be a mechanism for better correction of the error-prone synthesis catalyzed by DNA polymerase alpha.

FIGURE 1.

p300 binds and acetylates Dna2 both in vitro and in vivo. A, 2 μg of unmodified hDna2 (wtDna2) and ¹⁴C-acetylated in vitro hDna2 (AcDna2) was separated on a 4–15% SDS-polyacrylamide gel. The upper panel shows the Coomassie stain of the separated proteins and acts as a loading control for the unmodified and modified Dna2. The same gel was subsequently exposed to x-ray film for 1 week, and the acetylation status of Dna2 was analyzed (lower panel). B, 1 ng of purified hDna2 was incubated with 1 ng of recombinant catalytic domain (amino acids 1284–1673), 1 ng of recombinant catalytic domain (amino acids 965–1810), or 1 ng of recombinant full-length p300 in a binding assay. The bound proteins were immunoprecipitated (IP) with antibody against full-length p300, separated by gel electrophoresis, and Dna2 was detected by Western blotting (IB) with antibody against Dna2. C, HeLa whole cell extracts were used either to immunoprecipitate p300 and immunoblot for p300, Dna2, and FEN1 or immunoprecipitate Dna2 and immunoblot for p300, Dna2, and FEN1. The co-immunoprecipitated proteins and their molecular weights are indicated in the gel. ML, molecular weight ladder. D, HeLa whole cell extracts from either untreated or UV-treated cells were used to immunoprecipitate Dna2 and immunoblot for Dna2 and acetylated lysines (Ac-K). The proteins associated with the acetylated lysines detected by Western blotting were confirmed by stripping the blot and reprobing it with either anti-Dna2 or anti-FEN1 to confirm the detected protein.

FIGURE 2.

Nuclease activity of Dna2 is stimulated upon acetylation by p300. 5′–3′ endonuclease activity. A 53-nt flap substrate labeled at the 3′ end of the downstream primer was used to measure the 5′–3′ endonuclease activity of hDna2. Reactions containing 5 fmol of substrate and increasing concentrations (10, 20, and 50 fmol) of Dna2 alone, Dna2 and p300, and Ac-Dna2 were incubated for 10 min at 37 °C. A control reaction with 5 fmol of substrate and 10 fmol of FEN1 was used to identify the base of the flap. The labeled substrate is depicted above the gel with the asterisk indicating the site of the ³²P label. The substrate alone, 5′–3′ Dna2 cleavage products, FEN1 cleavage product, and cleavages beyond the base of the flap are indicated beside the gel.

FIGURE 3.

Ac-Dna2 shows increased ATPase and helicase activities. A, DNA-dependent ATPase activity of 500 fmol of unmodified and Ac-Dna2 was analyzed as described under “Experimental Procedures.” The ATPase activity of the unmodified and Ac-Dna2 is plotted in the graph. A control without DNA is included. B, helicase activity was assayed as described under “Experimental Procedures.” Reactions containing 1 fmol of M13-HPR partial duplex substrate and 1000 fmol of nuclease-defective hDna2-D294A alone (lanes 3 and 4), hDna2-D294A and p300 (lanes 5 and 6), and Ac-Dna2-D294A (lanes 7 and 8) were incubated for 15 min at 37 °C. Lane 1 represents the boiled substrate, and lane 2 represents the annealed M13-HPR partial duplex substrate. The labeled substrate and helicase products are indicated at the side of the gel. Positions of the substrate, helicase products, and nuclease products are indicated on the left of the figure.

FIGURE 4.

Increased binding efficiency of Ac-Dna2. Substrate binding efficiency of Dna2 and Ac-Dna2 was studied using electromobility gel shift assay. Five fmol of 53-nt substrate was incubated with increasing concentrations (10, 20, and 50 fmol) of Dna2 alone, Dna2 and p300, and Ac-Dna2, and the reactions were incubated for 10 min at room temperature and separated on a 5% polyacrylamide gel. The labeled substrate is depicted above the gel with the asterisk indicating the site of the ³²P label. The substrate alone and complexes containing Dna2-bound substrate are indicated beside the gel at the right.