1. Volume 10, Issue 5, Pages 1201-1211 (November 2002)
Flap Endonuclease 1 Efficiently Cleaves Base Excision Repair and DNA Replication Intermediates Assembled into Nucleosomes 
Christine F Huggins, David R Chafin, Sayura Aoyagi, Leigh A Henricksen, Robert A Bambara, Jeffrey J Hayes 
Molecular Cell 
Volume 10, Issue 5, Pages (November 2002)
DOI: /S (02)

2. Figure 1 Flap Substrate Preparation
(A) Preparation of nucleosome and naked DNA flap substrates. The indicated oligonucleotides (numbers) were annealed in the steps shown as described in the Experimental Procedures. The star indicates the position of radioactive label.
(B) Sequence detail of noncomplementary 5 nt flap. The sequence is numbered according to 5S gene start site of transcription (see Experimental Procedures). The arrows indicate the peaks of hydroxyl radical cleavage, where the DNA backbone is oriented maximally away from the histone octamer. The predicted initial FEN1 cleavage site is indicated by the middle arrow, top strand. The continuous flap strand is italicized.
(C) Flap substrates are assembled into canonical nucleosomes. Nucleosomes were reconstituted with control (native) and flap-containing DNA fragments labeled internally and analyzed by agarose nucleoprotein gel analysis. Lanes 1–4 contain the flap DNA substrate, flap nucleosomes, control DNA fragment, and control nucleosomes, respectively.
(D) Nucleosomes reconstituted with flap DNA substrates exhibit homogeneous translational positioning. Nucleosomes prepared as in (C) were analyzed on 5% acrylamide translational gels. Lane 1, control DNA; lane 2, control nucleosomes assembled with native 154 bp 5S DNA fragment; lane 3, naked 154 bp flap DNA stubstrate; lane 4, EcoRV-digested naked flap DNA substrate; lanes 5 and 7, reconstituted 154 bp flap DNA substrate; lanes 6 and 8, gradient-purified nucleosomes from the reconstituted products shown in lanes 5 and 7, respectively. Note that samples in lanes 5/6 and 7/8 were reconstituted with the after-displacement BER substrate and the 5 nt noncomplementary flap substrate, respectively.
(E) Presence of the flap does not alter the rotational position of DNA in reconstituted nucleosomes. Nucleosomes were reconstituted with either native 5S DNA or the noncomplementary 5 nt flap substrate radioactively labeled at the terminal phosphate of the bottom strand and were analyzed by hydroxyl radical footprinting as described in the Experimental Procedures. Lanes “DNA” and “Con” contain the hydroxyl radical cleavage products from digestion of native naked 5S DNA or native 5S nucleosomes. Lane “Flap” contains the products of hydroxyl radical cleavage of the 5 nt flap-containing nucleosomes. Lane G, Maxim-Gilbert G-reaction marker. The locations of the nucleosome dyad and of the flap are as indicated (arrows) while the dots correspond to the centers of the cleavage peaks.
Molecular Cell  , DOI: ( /S (02) )

3. Figure 2
FEN1 Preferentially Cleaves the Nucleosome Substrate at Subsaturating Enzyme Concentrations
(A) FEN1 enzyme titration. Nucleosomes reconstituted with the noncomplementary 5 nt flap substrate and the EcoRV-cleaved naked DNA substrate were mixed together in the same tube, and then digested with increasing amounts of FEN1 for 10 min as described in the Experimental Procedures. Digestion products were separated by sequencing gel electrophoresis analyzed by PhosphorImager. Lanes 1–6 contain 0, 0.01, 0.1, 1, 10, or 100 nM FEN1, respectively. Bands corresponding to the 92 nt and 48 nt undigested nucleosomal (Nuc) and naked DNA (DNA) substrates, respectively, are indicated as are the FEN1 digestion products from each (vertical bars). Quantification of the amount of nucleosomal and naked DNA substrate cleaved is shown at the bottom of the gel.
(B) The rate and extent of substrate cleavage by FEN1 increases with FEN1 concentration. The noncomplementary 5 nt nucleosomal and naked DNA substrates were incubated with FEN1, and the extent of digestion was determined at 0, 0.25, 0.5, 1, 3, 5, and 10 min after the addition of enzyme. Lanes 1, 8, 15, and 22 contain no FEN1. Products shown in lanes 2–7, 9–14, 16–21, and 23–28 were digested with 1, 2, 3, and 5 nM FEN1.
(C) The initial rate and total substrate cleavage is dependent on enzyme concentration. FEN1 cleavage was quantitated from gels such as those shown in (B) and plotted as the percent of original full-length substrate cleaved versus time. The left and right panels show the digestion profile of the nucleosomal and naked DNA substrates, respectively. Diamonds, triangles, circles, and squares depict digestions with 1, 2, 3, and 5 nM FEN1, respectively. The plots are the average of at least two independent experiments with error bars showing the standard deviations.
Molecular Cell  , DOI: ( /S (02) )

4. Figure 3
Preferential Cleavage of the Nucleosome by FEN1 Requires the Histone Tail Domains
Nucleosomes were reconstituted with the 5 nt noncomplementary flap substrate and either native (WT) or core histones lacking their N-terminal tail domains (Tailless).
(A) Nucleoprotein gel analysis of nucleosomes. Lane 1, free 154 bp noncomplementary flap-containing DNA template; lane 2, EcoRV-digested template; lanes 3 and 4, gradient-purified nucleosomes assembled with native or tailless core histones, respectively.
(B) The extent of digestion of native and tailless nucleosomes by FEN1 was determined after 2 min incubation as described in the Experimental Procedures and plotted as percent of uncut template remaining versus FEN1 concentration.
Molecular Cell  , DOI: ( /S (02) )

5. Figure 4
FEN1 Exhibits Cleavage Specificity for a Two-Flap Substrate within a Nucleosome
(A) Alternative conformations of the complementary flap substrate. The fully extended 5′ flap (upper left) can equilibrate to a structure with a smaller 5′ flap and a 3′ flap (lower left and lower right) and ultimately to a fully extended 3′ flap conformer (upper right). (Note that the bottom strand remains fully annealed in all intermediates.)
(B) FEN1 cleavage of nucleosomal two-flap substrate. Nucleosomes were reconstituted either with the complementary (Comp) or noncomplementary (Non-Comp) 5 nt flap substrates, mixed with the appropriate naked DNA template, and then digested with FEN1. Products of digestion were analyzed as above. Lanes 1–5 and 6–10 contain complementary and noncomplementary flap substrates, respectively. FEN1 was added to reactions to final concentrations of 0, 0.1, 1, 10, or 100 nM, as indicated by the wedge. The arrows indicate the initial site of endonucleolytic cleavage by the enzyme.
Molecular Cell  , DOI: ( /S (02) )

6. Figure 5 FEN1 Cleaves BER Intermediates Assembled into Nucleosomes
Nucleosomes reconstituted with either the before-displacement or after-displacement substrates (bottom) were mixed with the appropriate naked DNA control and digested with increasing amounts of FEN1, and the products were analyzed as described. In each set, FEN1 concentrations were 0, 0.1, 1, 10, or 100 nM in lanes 1–5, respectively, while lane 6 contains only the EcoRV-cleaved naked DNA substrate. The triangle indicates the AP site.
Molecular Cell  , DOI: ( /S (02) )

7. Figure 6
Sequential FEN1 and hDNA Ligase 1 Activity within the Same Nucleosome
Nucleosomes containing the complementary 5 nt flap substrate were incubated with FEN1 and hDNA ligase I, and the products were analyzed on sequencing gels as described in the Experimental Procedures. Lane 1, 154 nt marker; lanes 2–5, nucleosome substrate incubated with no enzymes, FEN1 alone, ligase alone, and FEN1 and ligase together, respectively. Lanes 6–9 are as in 2–5 except the samples were incubated for an additional 15 min with EcoRV to cleave any nonnucleosomal DNA present in the reaction. Symbols along the gel indicate, from top, bands in lane 9 corresponding to the fully ligated nucleosomal product, ligated and EcoRV-cleaved naked DNA, the original nucleosome substrate, FEN1-digested substrate, EcoRV-cleaved undigested substrate, and EcoRV-cleaved, FEN1-digested substrate.
Molecular Cell  , DOI: ( /S (02) )