1. Volume 7, Issue 6, Pages 1221-1231 (June 2001)
Regulation of Human Flap Endonuclease-1 Activity by Acetylation through the Transcriptional Coactivator p300 
Sameez Hasan, Manuel Stucki, Paul O Hassa, Ralph Imhof, Peter Gehrig, Peter Hunziker, Ulrich Hübscher, Michael O Hottiger 
Molecular Cell 
Volume 7, Issue 6, Pages (June 2001)
DOI: /S (01)

2. Figure 1 Fen1 Forms a Complex with p300
(A) p300 was immunoprecipitated from 150 μg HeLa nuclear extracts. Coimmunoprecipitated proteins were visualized by Western blot analysis using an anti-p300 antibody (top) or an anti-Fen1 antibody (bottom).
(B) Fen1 was immunoprecipitated in the presence or absence of ethidium bromide (Et. Br.).
(C) Cells were synchronized in G1 by serum starvation, in G1/S by addition of hydroxyurea, or in S phase by release thereof. Fen1 was immunoprecipitated and bound p300 was visualized by Western blot analysis
Molecular Cell 2001 7, DOI: ( /S (01) )

3. Figure 2 Mapping of the Fen1 Interaction Domain in p300
(A) Schematic representation of p300 and its interaction domains: netted box, cysteine-histidine rich region 1; dotted box, cysteine-histidine rich region 2; gray box, histone acetyltransferase (HAT) domain; and square-patterned box, cysteine-histidine rich region 3.
(B) Coomassie-stained SDS-PAGE gel of bacterially expressed and purified GST p300 fragments 1–5.
(C) GST pull-down experiments of Fen1 with bacterially expressed GST p300 fragments 1–5. Glutathione sepharose purified GST p300 fragments were incubated with bacterially expressed Fen1 (500 ng). Interacting Fen1 was visualized by Western blot analysis using an anti-Fen1 antibody.
(D) GST pull-down experiments of Fen1 in the presence of PCNA. Interacting Fen1 was visualized by Western blot analysis
Molecular Cell 2001 7, DOI: ( /S (01) )

4. Figure 3 Mapping of the p300 Interaction Region within Fen1
(A) Schematic view of Fen1 and its conserved domains. The N region ( ) and I region ( ) are catalytically conserved domains. The PCNA-interaction motif () is located adjacent to the basic C-terminal tail ( ).
(B) Precipitation experiments of wild-type and mutant Fen1 with immunoprecipitated p300. p300 was immunoprecipitated from HeLa nuclear extract and subsequently incubated with purified wild-type Fen1, ΔC Fen1, and ΔP Fen1, respectively. After extensive washing interacting proteins were visualized by Western blot analysis using an anti-Fen1 antibody.
(C) GST pull-down experiments of wild-type and ΔC Fen1 with bacterially expressed GST p300 fragments 4–5 and GST. Glutathione sepharose purified GST p300 fragments were incubated with purified wild-type and ΔC Fen1. Interacting Fen1 was visualized by Western blot analysis using an anti-Fen1 antibody.
(D) Immunoprecipitations of p300 in extracts of 293T cells expressing either myc-tagged wild-type Fen1 or myc-Fen1 ΔC. Bound proteins were visualized using Western blot analysis using an anti-p300 antibody (top) or an anti-myc antibody (lower panel)
Molecular Cell 2001 7, DOI: ( /S (01) )

5. Figure 4 Fen1 Is Acetylated In Vitro by p300
(A) p300 was immunoprecipitated from HeLa nuclear extracts and subsequently used in an acetylation assay with wild-type Fen1 and ΔC Fen1. As a control, an immunoprecipitation using an unspecific control antibody was performed. The lower panel showing a Coomassie-blue stained gel after the acetylation assay served as the input loading control. The same gel was subsequently analyzed using autoradiography (top panel).
(B) GST-p300-HAT or GST (control) were bacterially expressed and purified using glutathione sepharose and subsequently used in an acetylation assay with Fen1. The lower panel shows a Coomassie-stained gel after the acetylation assay, which was subsequently analyzed using autoradiography (top panel).
(C) Comparative acetylation of Fen1 and core histones. Seven micrograms of Fen1 and core histones were incubated with [14C]-acetyl-CoA in the presence or absence of p300 or GST-p300-HAT (upper and lower panels, respectively). The [14C]-acetate incorporation was measured by excising the radioactive band from the gel followed by analysis in a scintillation counter
Molecular Cell 2001 7, DOI: ( /S (01) )

6. Figure 5
Fen1 Is Acetylated In Vivo and This Acetylation Is Stimulated upon UV Treatment of the Cells
(A) The antibody raised against acetylated lysine residues is specific for acetylated Fen1. Seven μg of Fen1 was acetylated using immunoprecipitated p300 or using precipitates of an unspecific control antibody. The specificity of the antibody was verified by Western blot analysis. The upper panel shows a Western blot analysis for Fen1 as a control; the lower panel shows a Western blot analysis for acetylated proteins.
(B) Myc-tagged Fen1 was transiently overexpressed in 293T cells together with p300. Nuclear extracts of either empty vector, myc-Fen1, or myc-Fen1 ΔC transfected 293T cells were prepared and subsequently used for immunoprecipitation with an anti-myc antibody. Bound Fen1 was analyzed for in vivo acetylation using Western blot analysis. The top panel shows a Western blot against Fen1. The lower panel shows the same blot probed with an anti-acetylated lysine antibody.
(C) Nuclear extracts of empty-vector transfected, myc-Fen1 transfected, or myc-Fen1 transfected and UV treated 293T cells were prepared and subsequently used for immunoprecipitation with an anti-myc antibody. The top panel shows a Western blot against Fen1. The lower panel shows the same blot used in a Western blot analysis with an anti-acetylated lysine antibody.
(D) Acetylation of Fen1 does not influence its interaction with PCNA. Acetylated Fen1 and unmodified Fen1 were bound to Nickel-NTA beads used to pull down PCNA. Bound PCNA was visualized using Western blot analysis
Molecular Cell 2001 7, DOI: ( /S (01) )

7. Figure 6
Acetylation of Fen1 by GST-p300-HAT Significantly Reduces Fen1 Nuclease Activity
(A) Fifteen nanograms of purified Fen1 alone or in the combination with GST-p300-HAT and AcCoA as indicated on top of the panel were preincubated in HAT reaction buffer and subsequently added to 50 fmol of labeled DNA flap substrate (top of [B]) in Fen1 reaction buffer. The reaction products were resolved on a 15% denaturing polyacrylamide gel and visualized by autoradiography. The lower panel shows quantification of the results.
(B and C) Top: schematic view of the DNA substrates used for the flap endonuclease and 5′ → 3′ exonuclease assays. Upper panel: Fen1 was treated with GST-p300-HAT in the presence or absence of AcCoA and subsequently titrated to 50 fmol labeled flap substrate (B) or exonuclease template (C). Reaction time was 15 min at 30°C. The amounts of Fen1 added were 2.5, 5, 10, and 20 ng, respectively. The lower panel shows quantification of the results.
(D) Two nanograms of purified Fen1, 50 ng of purified PCNA, or a combination of both were added to 50 fmol of labeled DNA flap substrate (top of B) in Fen1 reaction buffer, containing 125 mM NaCl. Lane 1 represents the buffer control. The reaction products were resolved on a denaturing polyacrylamide gel and visualized by autoradiography. The lower panel shows quantification of the results.
(E) Upper panel: Fen1 was treated with GST-p300-HAT in the presence or absence of AcCoA and subsequently titrated to 50 fmol labeled flap substrate in the presence of 125 mM NaCl and 50 ng of PCNA. Reaction time was 15 min at 30°C. The amounts of Fen1 added were 0.25, 0.5, 1, and 2 ng, respectively. The lower panel shows quantification of the results.
(F) Upper panel: 10 ng of Fen1 were treated with GST-p300-HAT in the presence or absence of AcCoA and subsequently added to 250 fmol labeled flap substrate in the presence of 125 mM NaCl and in the presence or absence (as indicated on top) of 250 ng of PCNA. Reaction mixtures were incubated at 30°C and aliquots were taken after 0, 5, 10, 15, and 30 min, respectively. Products were separated on a gel. The lower panel shows quantification of the results
Molecular Cell 2001 7, DOI: ( /S (01) )

8. Figure 7
Influence of Acetylation on the DNA Binding Activity of Fen-1 and Identification of Acetylated Lysine Residues
(A) Acetylated Fen1 has weaker DNA binding activity. DNA binding activity of Fen1 was investigated by EMSA using unacetylated or acetylated Fen1.
(B) The masses of tryptic peptides derived from nonacetylated (upper panel) and acetylated (lower panel) of Fen1 were determined by MALDI-TOF mass spectrometry. Schematic diagram of identified acetylated lysine residues within Fen1
Molecular Cell 2001 7, DOI: ( /S (01) )