Regulation of Human Flap Endonuclease-1 Activity by Acetylation through the Transcriptional Coactivator p300

Molecular Cell, Vol. 7, 1221–1231, June, 2001, Copyright 2001 by Cell Press

Regulation of Human Flap Endonuclease-1 Activity by Acetylation through the Transcriptional Coactivator p300 Sameez Hasan,1,4 Manuel Stucki,1,4,5 Paul O. Hassa,1 Ralph Imhof,1 Peter Gehrig,2 Peter Hunziker,2 Ulrich Hu¨bscher,1 and Michael O. Hottiger1,3 1 Institute of Veterinary Biochemistry 2 Institute of Biochemistry University of Zu¨rich Winterthurerstrasse 190 8057 Zu¨rich Switzerland

Summary We describe a role for the transcriptional coactivator p300 in DNA metabolism. p300 formed a complex with flap endonuclease-1 (Fen1) and acetylated Fen1 in vitro. Furthermore, Fen1 acetylation was observed in vivo and was enhanced upon UV treatment of human cells. Remarkably, acetylation of the Fen1 C terminus by p300 significantly reduced Fen1’s DNA binding and nuclease activity. Proliferating cell nuclear antigen (PCNA) was able to stimulate both acetylated and unacetylated Fen1 activity to the same extent. Our results identify acetylation as a novel regulatory modification of Fen1 and implicate that p300 is not only a component of the chromatin remodeling machinery but might also play a critical role in regulating DNA metabolic events. Introduction p300 and its homolog CBP were identified as transcriptional coactivators which are fundamentally important in various signal-modulated transcriptional events (Eckner et al., 1994). Both proteins have been shown to interact with a diverse set of sequence-specific transcription factors that participate in a broad spectrum of biological activities (Snowden and Perkins, 1998). The ability of p300/CBP to enhance transcription is believed to be accomplished in two modes: first, by acting as a bridging factor, thus recruiting the RNA polymerase II holoenzyme via interaction with general transcription factors; and second, by acetylation of histones via its histoneacetyl-transferase (HAT) domain. Acetylation of histones leads to an “open chromatin” structure, which is sought to facilitate binding of transcription factors to DNA (Utley et al., 1998). Mouse embryos lacking both p300 alleles died around midgestation showing pleiotropic defects in morphogenesis, in cell differentiation, and in proliferation as a result of a reduced ability to synthesize DNA (Yao et al., 1998). The reduced ability of p300⫺/⫺ fibroblasts to synthesize DNA could be explained by p300

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Correspondence: [email protected] These authors contributed equally to this work. 5 Present address: Wellcome/CRC Institute of Cancer and Developmental Biology, University of Cambridge, Tennis Court Road, Cambridge, CB2 1QR, United Kingdom. 4

being responsible as transcriptional coactivator for the activation of S-phase-specific genes, or by p300 itself being an important cofactor for DNA metabolism. To address this question, we investigated whether p300 would associate with proteins known to play an important role in DNA metabolism. We identified human Fen1 as a protein forming a complex with p300. Fen1 is a structure-specific metallonuclease with important roles in DNA metabolic events such as replication and repair (reviewed by Lieber, 1997). In DNA replication it participates in the removal of the displaced RNA-DNA primers during Okazaki fragment maturation (reviewed by Bambara et al., 1997). In repair, Fen1 might be involved in the removal of DNA base damages caused by base-damaging agents through participation in one or several excision repair pathways (Klungland and Lindahl, 1997). The relevance of these observations is underscored by in vivo data using yeast Fen1 null mutant yeast strains (Sommers et al., 1995; Vallen and Cross, 1995). These strains are generally viable indicating the presence of a redundant enzymatic activity in yeast. Genetic analysis of these strains revealed a pleiotropic phenotype including temperature-sensitive growth defect, sensitivity to UV radiation and methyl methane sulfonate (MMS) (Reagan et al., 1995), and elevated rates of mitotic recombination. Moreover, Fen1 has recently been proposed to be involved in nonhomologous end joining and mitotic gene conversion in yeast (Holmes and Haber, 1999; Wu et al., 1999). Vertebrate Fen1 is homologous to the Rad2 group of nucleases that contain three homology boxes named N-, I-, and C-regions (Harrington and Lieber, 1994). While the N- and I-regions are clearly involved in catalysis, a role for the C-region has not yet been determined. Interestingly this C-terminal region is not present in recently discovered archaea orthologs of Fen1, leading to the hypothesis that this highly basic region may contain a nuclear localization signal (Hosfield and Tainer, 1998). Recently, however, we found that this region is essential for substrate binding and might provide the enzyme with a function that can regulate enzymatic activity through modulation of the substrate binding step (Stucki et al., 2001). The biochemistry of Fen1 has been studied by several groups and the Fen1 crystal structures of two archaea orthologs are available (Hosfield et al., 1998; Hwang et al., 1998). The enzyme employs a unique cleavage mechanism for substrates containing single-stranded 5⬘ tails or flap structures. It cleaves the flap by recognizing the 5⬘ end, tracking the length of the tail and cleaving at the junction between double-stranded and single-stranded DNA (reviewed in Lieber, 1997). In vivo, Fen1 is complexed with the proliferating cell nuclear antigen (PCNA) via a consensus motif (reviewed in Jonsson and Hu¨bscher, 1997). Fen1 activity is stimulated by PCNA in vitro under physiological ionic conditions (Li et al., 1995; Wu et al., 1996). Besides PCNA, the DNA2 helicase/endonuclease has been identified to interact both genetically and physically with Fen1 in budding yeast (Budd and Campbell, 1997). Based on the biochemical properties of DNA2 and Fen1 and genetic results from yeast, an alternative

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Figure 1. Fen1 Forms a Complex with p300 (A) p300 was immunoprecipitated from 150 ␮g 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.

model for Okazaki fragment metabolism has been proposed (Bae and Seo, 2000; Kang et al., 2000). In this model, the sequential action of these two processing enzymes, DNA2 and Fen1, is required for the removal of primer RNA and DNA. Although this model appears to require redundant enzymatic functions, it is supported by many genetic and biochemical observations and would explain the viability of Fen1 deleted yeast mutant strains (Bae and Seo, 2000; Kang et al., 2000). In this study, we show that Fen1 exo- and endonuclease activity is inhibited in vitro upon acetylation of Fen1 by the p300 HAT domain. This inhibitory effect due to acetylation cannot be overcome by PCNA, although PCNA stimulates both acetylated and unacetylated Fen1 to the same extent. We show that Fen1 is modified by acetylation in vivo presenting evidence that an enzyme involved in DNA metabolism is modified by acetylation. Acetylation of Fen1 is markedly enhanced upon treatment of the cells with UV radiation. Together, these results implicate that p300 as a component of the chromatin remodeling complex not only induces chromatin changes but might have an additional role in regulating DNA metabolism.

Results Fen1 Forms a Complex with p300 To examine whether p300 would act as a cofactor for enzymes involved in DNA replication and repair, we performed immunoprecipitations of p300 from HeLa nuclear extracts and analyzed bound proteins by Western blot analysis using a specific anti-Fen1 antibody. Fen1 could be detected together with p300 in the bound protein fraction (Figure 1A). Experiments with a control antibody showed neither the presence of p300 nor of Fen1. Vice versa, endogenous p300 could be coimmunoprecipitated using a Fen1-specific antibody (Figure 1B, left panel). Coimmunoprecipitation of p300 was not mediated by DNA, because addition of ethidium bromide to the immunoprecipitated fraction did not affect p300/ Fen1 binding (Figure 1B, right panel). Immunoprecipitation experiments with cells arrested in different cellcycle states revealed that complex formation of p300/ Fen1 is not dependent on the cell cycle (Figure 1C). Cellcycle arrest was monitored using FACS analysis (data not shown). Together, these results suggest that p300 and Fen1 form a tight complex mediated through protein interactions throughout the cell cycle.

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pressing the amino acid residues 1197–1674 of p300, comprising the whole HAT domain of p300, and subsequent Western blot analysis of bound Fen1, revealed that Fen1 is also able to interact with the HAT domain, but to a weaker extent than with fragment 4 (data not shown). p300 binding of Fen1 could not be completed with a 5-fold molar excess of PCNA, a well documented Fen1 interacting protein (Figure 2D). Western blot analysis for PCNA revealed that Fen1 is able to bind simultaneously to p300 and PCNA (data not shown).

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, cysteinehistidine 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.

Mapping of the Fen1 Interaction Domain within p300 To confirm this interaction and to map the Fen1 interaction domain in p300, the latter was divided into five fragments which were subsequently cloned and expressed as GST fusion proteins (Figures 2A and 2B). GST pull-down experiments with these fragments were performed using bacterially expressed and purified histidine-tagged wild-type Fen1. Western blot analysis of the bound protein using a specific anti-Fen1 antibody indicated that fragment 4 of p300 is able to interact with Fen1 (Figure 2C), thus confirming that the interaction is not mediated by DNA. This fragment corresponds to amino acid residues 1459–1892 of p300 and contains part of the HAT domain (see also Figure 2A). These results indicate that Fen1 is likely to interact directly with p300 specifically through distinct domains located between amino acids 1459 and 1892. GST pull-down experiments with the GST-p300-HAT fusion protein ex-

Mapping of the p300 Interaction Domain in Fen1 To map the p300 interaction domain in Fen1, two deletion mutants were constructed. One mutant (⌬C) lacks the basic C-terminal tail of 20 amino acids that is characterized by a striking accumulation of basic residues (8 lysines and 1 arginine) and the other (⌬P) lacks the 8 amino acids containing the PCNA interacting motif (QGRLDDFF) adjacent to the C-terminal tail (Figure 3A) (Stucki et al., 2001). Wild-type and mutant Fen1 were bacterially expressed as histidine-tagged proteins and purified to homogeneity (data not shown). p300 was immunoprecipitated from HeLa cell extracts and subsequently incubated with either wild-type or one of the two Fen1 mutants. Bound proteins were analyzed by Western blot analysis using a specific Fen1 antibody. While wild-type Fen1 and the ⌬P mutant Fen1 were both able to bind to immunoprecipitated p300, no binding was observed with the ⌬C mutant (Figure 3B). GST pulldown experiments with the purified p300 fragments fused to GST confirmed that wild-type Fen1, but not the ⌬C mutant Fen1, was able to bind to fragment 4 (Figure 3C). The fact that bacterially expressed and purified proteins were able to interact suggests that the interaction of p300 and Fen1 is not mediated by other proteins. These results were verified using 293T cells transfected with plasmids harboring myc-tagged Fen1 or myctagged Fen1 ⌬C. Immunoprecipitations of p300 from nuclear extracts and subsequent analysis of bound Fen1 proteins by Western blot analysis using an anti-myc antibody showed that only wild-type Fen1 was able to form a complex with p300 but not the mutant (⌬C) lacking the C-terminal domain (Figure 3D). These results suggest that Fen1 can specifically interact with p300 through its C-terminal domain. Fen1 Is Acetylated In Vitro by p300 p300 is not only binding the histone acetyltransferase P/CAF (Cho et al., 1998), but also possesses intrinsic HAT activity (Ogryzko et al., 1996). We therefore investigated whether p300 could acetylate Fen1. p300 was immunoprecipitated from HeLa nuclear extract, purified from endogenous proteins by high salt washes, and subsequently used together with purified recombinant Fen1 in an acetylation assay. Fen1 was very potently acetylated by immunoprecipitated p300, whereas immunoprecipitations with a control antibody did not result in acetylation of Fen1 (Figure 4A, left panel). The same experiment with the C terminally truncated form of Fen1 (⌬C) resulted in no acetylation of Fen1 (Figure 4A, right panel). These results revealed that the Fen1 mutant (⌬C), which was no longer able to bind to p300 (see Figure 3B, middle panel), also cannot be acetylated (Figure

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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, ⌬C Fen1, and ⌬P 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 ⌬C Fen1 with bacterially expressed GST p300 fragments 4–5 and GST. Glutathione sepharose purified GST p300 fragments were incubated with purified wild-type and ⌬C 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 ⌬C. Bound proteins were visualized using Western blot analysis using an anti-p300 antibody (top) or an anti-myc antibody (lower panel).

4A). Coomassie blue staining of the gel confirmed that comparable amounts of Fen1 were used in both experiments (Figure 4A, bottom row). Next, bacterially expressed GST-p300-HAT and purified Fen1 were tested in a HAT assay. Fen1 was acetylated by GST-p300-HAT bound to glutathione beads to the same extent when compared to acetylation with immunoprecipitated p300. GST alone could not acetylate Fen1 (Figure 4B). Finally, acetylation of Fen1 by immunoprecipitated p300 (Figure 4C, upper panel) or by the bacterially expressed HAT domain of p300 (Figure 4C, lower panel) was compared to acetylation of core histones and was found to be comparable. Fen1 Is Acetylated In Vivo and This Acetylation Is Stimulated upon UV Treatment of Human Cells To test whether Fen1 is acetylated in vivo, we used an antibody raised against acetylated lysine residues. To verify the ability of the antibody to recognize only acetylated Fen1, the purified enzyme was acetylated in vitro by either p300 immunoprecipitated from HeLa extracts or a control sample, and acetylation was analyzed by Western blot analysis. The antibody was able to recognize specifically the acetylated form of Fen1, whereas the nonacetylated protein was not recognized (Figure

5A). To test whether Fen1 was acetylated in vivo, 293T cells were transfected with either an empty vector or with a vector expressing either myc tagged wild-type or mutated Fen1 (⌬C). After nuclear extracts preparation, myc-tagged Fen1 fusion proteins were immunoprecipitated using the anti-myc-specific antibody and subsequently analyzed by Western blot analysis using the anti-acetylated lysine antibody. Western blot analysis revealed that wild-type Fen1 but not the (⌬C) mutant is acetylated in vivo (Figure 5B, lower panel; compare lanes 2 and 4). Western blot analysis with the specific anti-Fen1 antibody revealed that the same amount of proteins are expressed and immunoprecipitated. Cotransfection of p300 into these cells resulted in a slight increase of Fen1 acetylation, though the expression and the amount of immunoprecipitated Fen1 remained constant (Figure 5B, compare lanes 2 and 3). This finding indicates that p300 is at least in part responsible for the observed acetylation of Fen1 in vivo. The fact that Fen1 ⌬C was not acetylated under the tested conditions (Figure 5B) supported the idea that acetylation of Fen1 in vivo was dependent on p300, because this mutant was no longer able to interact with p300. Since Fen1 deletion causes UV sensitivity in yeast (Reagan et al., 1995), we next investigated whether UV radiation would affect acet-

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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 ⌬C 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 Coomassiestained 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]-acetylCoA 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.

ylation of Fen1. Myc-Fen1 transfected cells were treated with 20mJ/m2 UV radiation, and the acetylation status of myc-Fen1 was analyzed by Western blot analysis after immunoprecipitations of Fen1 with the anti-mycspecific antibody. UV treatment of the cells revealed a 12-fold increase of Fen1 acetylation, compared to untreated cells (Figure 5C). This effect was not due to an

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 ␮g 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, mycFen1, or myc-Fen1 ⌬C 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.

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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

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increase of the amount of immunoprecipitated Fen1, because blotting of the same membrane with the Fen1specific antibody indicated that the same amount of Fen1 was immunoprecipitated (Figure 5C, upper panel). Furthermore, the expression level of Fen1 was found to be unaltered upon UV radiation (Warbrick, 1998). These results suggest that Fen1 is acetylated in vivo and that the acetylation is heavily increased upon treatment of the cells with UV radiation. Acetylation of Fen1 Does Not Influence Its Binding to PCNA or to p300 Next, we investigated whether acetylation of Fen1 would influence its binding to PCNA and to p300. Recombinant His-tagged Fen1 was acetylated in vitro by GST-p300HAT and subsequently bound to Ni-NTA beads. Charged beads were incubated with recombinant PCNA and bound PCNA subsequently assessed by Western blot analysis. Acetylation of Fen1 did not affect its binding to PCNA (Figure 5D) or to recombinant fragment 4 of p300 (data not shown). Acetylation of Fen1 by GST-p300-HAT Reduces Fen1 Activity Significantly To investigate whether acetylation would affect the overall nuclease activity of Fen1, purified human Fen1 was incubated with GST-p300-HAT either in the presence or absence of AcCoA and subsequently tested in a flap-endonuclease assay using three annealed oligonucleotides that form a linear, radioactively-labeled artificial flap structure as a template (top of Figures 6A and 6B). Fifteen nanograms of Fen1 hydrolyzed 70% of the flap DNA in 15 min (Figure 6A, lane 1). Addition of AcCoA and GST-p300-HAT alone had virtually no effect on the flap-endonucleolytic activity of Fen1 (lanes 2 and 3), confirming that bacterially expressed and purified GSTp300-HAT was not contaminated with any nuclease activity (lane 4). However, when 15 ng of Fen1 was preincubated with GST-p300-HAT in the presence of AcCoA, only 12% of the template was hydrolyzed in 15 min (Figure 6A, lane 5). In order to quantify the inhibitory effect of Fen1 acetylation, titrations of unacetylated and acetylated Fen1 were performed. Fen1 was preincubated with GST-p300-HAT (either in the presence or absence of AcCoA) and incubated with the artificial flap template (Figure 6B). After resolving the reaction products on a denaturing polyacrylamide gel, bands were quantified using a PhosphorImager. These data revealed a 5.7-fold inhibition of acetylated Fen1 versus the unacetylated enzyme. Since Fen1 was shown to possess also a 5⬘ → 3⬘ exonuclease activity which acts preferentially at nicks and, with lower efficiency, also at gaps or recessed 5⬘ ends on double-stranded DNA (Lieber, 1997), we next tested whether acetylation would affect the exonuclease activity of Fen1. The same assay

conditions were used as for the flap-endonucleolytic activity; but the substrate was replaced by three annealed oligonucleotides that together form a linear, double-stranded DNA containing a single nick (top of Figure 6C). Quantification of the released labeled mononucleotide revealed that acetylation of Fen1 results in a 6.3fold inhibition of the Fen1 exonucleolytic activity (Figure 6C). Endo- and exonucleolytic activities of Fen1 are inversely proportional to monovalent salt concentrations in the physiological range. It has been shown, however, that PCNA can stimulate Fen1 activity several-fold under physiological salt conditions (125 mM salt). Under these conditions, Fen1 is virtually inactive in the absence of its cofactor PCNA. Kinetic analysis revealed that PCNA enhances Fen1 binding to the flap structure and stabilizes this complex, thus increasing the cleavage efficiency of Fen1 (Tom et al., 2000). To efficiently stimulate Fen1 activity, the PCNA trimer must encircle the DNA and must be located “below” the flap (Jonsson et al., 1998). In our study, PCNA-dependent Fen1 activity was determined at 125 mM NaCl. Under these conditions, Fen1 alone was almost inactive (Figure 6D, lane 2). Addition of PCNA that is loaded spontaneously onto the linear flap DNA stimulated the nuclease activity of Fen1 about 10-fold (Figure 6D, lane 4). To determine whether PCNA stimulation of Fen1 was affected by the acetylation of Fen1, endonuclease experiments were repeated in the presence of 125 mM NaCl and an excess of PCNA. PCNA stimulates Fen1 seen by the amount of Fen1 used in the experiments but was not able to overcome the inhibitory effect of Fen1 acetylation (Figures 6E and 6F), despite the fact that it was able to bind to acetylated Fen1 (see Figure 5D). In contrary, quantitative analysis of the inhibitory acetylation effect revealed a 10.1-fold inhibition in comparison to 5.7-fold at low-salt condition and in the absence of PCNA. A time course experiment strengthened the obtained results indicating that the reaction time of 15 min is at the maximal level of substrate turnover and that the reaction was fully dependent on PCNA under the conditions used (Figure 6F). Acetylation of Fen1 Reduces Its DNA Binding Activity To investigate whether the observed reduced enzymatic activity of acetylated Fen1 was a result of reduced DNA binding activity, Fen1 was acetylated by GST-p300-HAT coupled to glutathione sepharose beads and subsequently tested in an electrophoresis mobility shift assay (EMSA). The results indicated that acetylation of Fen1 significantly reduced the binding activity of Fen1 (Figure 7A). Identification of Acetylated Lysine Residues in Fen1 Finally, either unacetylated or acetylated Fen1 was digested with trypsin and the resulting peptides were ana-

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.

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HAT (Figure 7B). All identified acetylated lysine residues were located at the C-terminal end, which was previously shown to be important for DNA binding (Stucki et al., 2001). Discussion

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.

lyzed by matrix-assisted laser desorption time-of-flight (MALDI-TOF) mass spectrometry (Figure 7B). All peptide masses found for the nonacetylated protein matched the theoretical masses of tryptic peptides of Fen1. For the acetylated protein six additional peptides, T1, T2, T3, T4, T5, and T6, were found, whose masses of their singly charged ions were 821.97, 977.47, 1019.44, 1047.45, 1636.58, and 1863.73, respectively. Four of these peptides were isolated by reversed-phase HPLC and sequenced by Edman degradation. The following sequences were obtained: T1: TGAAG(acK)FK; T4: VTGSLSSA(acK)R; T5: G(acK)LAAALEHHHHHH; T6: G(acK) G(acK)LAAALEHHHHHH. Their theoretical masses correspond to the measured ones. Peptides T2: TGAAG (acK)FKR or TGAAGKF(acK)R and T3: TGAAG(acK)F (acK)R could not be isolated as single fractions by HPLC, but their sequences could be identified by the good agreement of their theoretical masses of 977.55 and 1019.56 with the measured masses. Together these experiments suggested that lysines 354, 375, 377, and 380 of the Fen1 sequence were acetylated by GST-p300-

Here we report that the human structure-specific replication- and repair-nuclease Fen1 forms a complex with the transcriptional coactivator p300. This interaction could be confirmed with bacterially expressed and purified Fen1 and p300 fragments indicating that the interaction is direct. The interaction was not mediated by DNA, since addition of ethidium bromide had no effect on the ability of Fen1 to coimmunoprecipitate with p300. Amino acids 360–380 at the C terminus of Fen1 and the amino acid residues 1459–1892 of p300 were responsible for the interaction of Fen1 with p300. In addition, we found that Fen1 is an acetylated protein in vivo and that Fen1 can be modified by the histone acetyl transferase activity of p300 in vitro at the C terminus, thus identifying a novel target of the acetyltranferase activity of p300. Investigations of the biochemical consequences of Fen1 acetylation by p300 revealed that the acetylation of Fen1 reduces its endo- and exonuclease activity in vitro. Both endo- and exonuclease activity of Fen1 were inhibited to the same extent after acetylation by the HAT domain of p300, although the overall substrate turnover was about eight times lower in the case of the nick substrate (exonuclease activity). This is not surprising, because it is well established that Fen1 has lower exo- than flapendonuclease activity (Lieber, 1997). PCNA was able to stimulate both unacetylated and acetylated Fen1 to the same extent, although the acetylated Fen1 showed a severely reduced enzymatic activity indicating that the acetylation does not affect Fen1 binding to PCNA but rather decreases the affinity to the substrate DNA as confirmed by EMSA. UV treatment of cells results in a significant increase of Fen1 acetylation, as the acetylation of p53 by p300/ CBP was shown to be similarly stimulated by UV treatment (Liu et al., 1999). The molecular nature of UV stimulated acetylation is currently not known. Different scenarios can be foreseen. The introduction of DNA lesions by UV radiation might activate other protein modifying enzymes such as kinases (Sakaguchi et al., 1998) that enhance acetylation of the target protein by p300 due to phosphorylation. This was shown to be the case for p53 (Sakaguchi et al., 1998). Simultaneously certain acetylases could be stimulated upon UV treatment, which would result in an increase of overall acetylated sites on proteins. Alternatively activated deacetylases could be inhibited upon UV treatment, which would also lead to an overall increase of the acetylated sites. It is uncertain whether p300 is responsible for the acetylation of Fen1 in vivo. Several lines of evidence indicate however that p300 plays a critical role in this process. Acetylation of Fen1 in vivo could be increased by overexpression of p300. The p300 interacting domain of Fen1 was mapped to be located at the C terminus and consequently the Fen1 (⌬C) mutant was not acetylated in vivo. Finally, given that p300 and Fen1 can be found in a complex suggests that acetylation by either p300 or a p300-associated acetylase is very likely.

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We identified 4 lysine residues within Fen1 which could be acetylated by the HAT domain of p300 in vitro. They lie in the basic tail of 36 amino acids at the very C terminus of Fen1, adjacent to the PCNA binding motif (Warbrick, 1998). No structural data about this domain is available, because it is absent in the solved X-ray structures of the archae Fen1 homologs (Hosfield and Tainer, 1998). We found that a deletion of the C-terminal tail results in a protein unable to interact with or be acetylated by p300 under physiological conditions. Enzymatic characterization of this Fen1 (⌬C) mutant revealed that it is significantly less active, in comparison to wild-type Fen1 in vitro, due to a severe defect in substrate binding (Stucki et al., 2001), demonstrating that the C terminus is essential for substrate binding. Interestingly, most lysines within this C-terminal tail are conserved in eukaryotes indicating that acetylation of these four residues might be an evolutionarily conserved regulatory mechanism within higher eukaryotes. Our results suggest that the C-terminal domain of Fen1 contains two functionally distinct regions, one PCNA interaction motif and one basic C-terminal tail that is essential for substrate binding which can be acetylated. Both regions are important for Fen1’s catalytic mechanism, albeit not necessarily at the same level. Possibly the C-terminal tail is responsible for general DNA binding activity whereas PCNA brings the catalytic center of Fen1 into the “right position” to perform the hydrolysis reaction. p300/CBP have not only been shown to acetylate histones (Ogryzko et al., 1996) but also nonhistone proteins such as p53 (Gu and Roeder, 1997), E2F (Martinez-Balbas et al., 2000), and HIV Tat (Kiernan et al., 1999), thereby regulating the activities of these proteins by modulating either their affinity to DNA or to other proteins. Acetylation of E2F increased its affinity to DNA thereby enhancing the transcriptional activation by E2F in vivo (Martinez-Balbas et al., 2000). Also, in the case of p53, an increased DNA binding activity upon acetylation was observed and explained with a conformational change of the regulatory C-terminal domain of the protein upon acetylation, which leads to a structure with higher DNA binding activity (Gu and Roeder, 1997). In contrast, our findings indicate that acetylation of Fen1 severely affects enzymatic activity as a result of reduced DNA binding activity. A similar effect was observed when Tat was acetylated by p300, which promoted its dissociation from the TAR RNA (Kiernan et al., 1999). Together these observations would support the hypothesis that the reduced activity of acetylated Fen1 is the result of a reduced substrate binding capacity due to the loss of essential positive charges at the C terminus. It is not clear at present whether this mode of regulation can simply be explained by an overall increase of negative charges within the Fen1 protein upon acetylation (which would decrease its DNA binding activity) or whether a conformational change takes place that makes the active site less accessible. The inhibitory effect of Fen1 acetylation on its enzymatic activity is puzzling, considering that Fen1 is an important nuclease in different DNA metabolic processes. The most likely explanation for this paradoxical observation is based on an alternative route for Okazaki fragment processing that has recently emerged, involv-

ing the action of the essential DNA2 helicase/endonuclease (Bae and Seo, 2000; Kang et al., 2000). In this model, DNA synthesis extending the 3⬘ end of an Okazaki fragment runs into the 5⬘ end of the next Okazaki fragment and displaces it. Normally, as soon as this flap length is a few nucleotides, it would be a well-suited target for hydrolysis by the flap endonuclease activity of Fen1 (Harrington and Lieber, 1994). In case of Fen1 inhibition, there would be more extensive strand-displacement synthesis, leading to the displacement of the entire primer-DNA that was synthesized by the error prone DNA polymerase ␣-primase complex (pol ␣). In such a case, the resulting single-stranded region exposed by the displaced flap is a substrate for the single-strand binding replication protein A (RP-A). Indeed, recent data revealed the importance of RP-A in mediating the sequential and ordered action of the two endonucleases DNA2 and Fen1 during Okazaki fragment processing (Y.-S. Seo, personal communication). RP-A combines with single-stranded DNA by forming a stable complex with at least 30 nucleotides (Kim et al., 1992). Strikingly, this is approximately the length of initiator DNA synthesized by pol ␣ before replication factor C abrogates primer synthesis and induces a polymerase switch from primer synthesis by pol ␣ to the processive elongation by DNA polymerase ␦ containing an intrinsic proofreading 3⬘ to 5⬘ exonuclease function (Mossi et al., 2000). It is conceivable that downregulation of Fen1 by acetylation is thought to prevent premature Okazaki fragment processing by Fen1. This would guarantee the full removal of the initiator DNA synthesized by pol ␣, which would then be a significant advantage for cells to maintain genomic integrity since this mechanism is independent of a mismatch correcting system that removes any errors inserted in the primer RNA/DNA. Yet it is still possible that there might be different pools of Fen1 involved in different processes within the cells that are dependent on their context to be sensitive to acetylation. Fen1-knockout strains were shown to be hypersensitive to UV radiation and MMS treatment, but not to ␥-radiation (Reagan et al., 1995), suggesting that Fen1 is either directly involved in the excision repair of induced DNA damages or indirectly functions in a DNA damage-tolerance mechanism. Interestingly, mutations in the yeast Fen1 allele cause increased rates of mitotic crossing over and are lethal in combination with mutations in genes that are involved in homologous recombination (Symington, 1998). This suggests that lesions generated in the absence of Fen1 activity must be processed by homologous recombination. The same might be true when Fen1 activity is downregulated by acetylation after UV damage, which may trigger recombination repair, which is an error-free repair pathway. Consistent with this idea is the observation that only Fen1 from diploid organisms being able to maintain genomic integrity via homologous recombination includes the C-terminal basic domain, whereas Fen1 from haploid organisms (Archaea) lacks this domain. Through the interaction with Fen1, p300 might be involved not only in the regulation of Fen1 activity, but also in the regulation of DNA metabolic events in general. p300 as a protein with intrinsic histone acetyltransferase activity (Ogryzko et al., 1996) and as a component

Molecular Cell 1230

of a chromatin remodeling complex (Cho et al., 1998) might change the structure of the chromatin adjacent to specific DNA sites, inducing chromatin changes that facilitate Fen1 function. An enhanced chromatin remodeling machinery might therefore be necessary to facilitate the access to DNA of the DNA synthesis machinery (Hasan et al., 2001). p300 might thus be an example of a protein that is well known to be involved in gene activation but might also play a critical role in regulating DNA metabolic events. Taken together, our results indicate that p300 might play a critical in the role of regulation of DNA metabolic events via its interaction with Fen1 and furthermore by regulating Fen1 nuclease activity upon acetylation. Experimental Procedures Plasmids p300 fragments 1–5 corresponding to amino acids 1–672, 672–1193, 1069–1459, 1459–1892, and 1893–2414 are described in Hasan et al. (2001). GST-p300-HAT (amino acids 1197–1674) was amplified using PCR and cloned into pGex6P2. The primers used were 5⬘-CTCGATCGTCGACAGGTATCATTTCTGTGAGAAGTG-3⬘ and 5⬘-GATCGAGGTCGACTCACTTGCATTCATTGCAGGTGTAG-3⬘. Proteins Human PCNA was produced in E. coli using the plasmid pT7/hPCNA and purified to homogeneity as described (Schurtenberger et al., 1998). Human Fen1 cDNA cloning and the generation of the ⌬C mutant (amino acids 360–380) and the ⌬P mutant (amino acids 337– 344) are described in Stucki et al. (2001). GST fusion proteins were expressed in E.coli and purified as described in Hasan et al. (2001). Immunoprecipitation Immunoprecipitations were performed as described (Hasan et al., 2001) with 1 ␮g of an anti-p300 antibody (PharMingen, 14991A), 1 ␮g of an anti-Fen1 antibody, or a control IgG (Sigma, M9269).

acetylated versus the nonacetylated Fen1 protein. Candidate peptides were separated on a C18 column (Vydac, 1 mm [inner diameter] ⫻ 250 mm) using a linear gradient of acetonitrile in 0.1% (v/v) TFA at a flow rate of 50 ␮l/min. The masses of the peptides were determined by directing 10% of the effluent to an electrospray mass spectrometer (Sciex, API III⫹). The remaining 90% were collected and used for Edman degradation on a Model G1005A protein sequencer (Agilent Technologies). Acetylated lysine residues were unambiguously identified as PTH-acLys, which elutes just after PTH-Ala. Electrophoresis Mobility Shift Assays (EMSA) Fen1 was acetylated as described above and EMSA was performed as described (Stucki et al., 2001) using 250 ng of Fen1 and 50 fmol of labeled template. Fen1 Assay Fen1 assays were conducted as described (Stucki et al., 2001). The activity assays were performed by preincubating purified Fen1 together with glutathione sepharose bound GST-p300-HAT and AcCoA (as indicated in the figures) in the described reaction. Acknowledgments We thank A. Dutta (Harvard Medical School) for the gift of the plasmid harboring wild-type and mutant myc-Fen1, E. Warbrick (University of Dundee) for the Fen1 antiserum, Y. Seo (Sungkyunkwan University) for sharing unpublished results, F. Ka¨lin for technical assistance, and members of the Institute of Veterinary Biochemistry (University of Zu¨rich) for their helpful advice and discussions. This work was supported in part by the Swiss National Science Foundation program 3100-57285.99 to S.H. and P.O.H. and program 310043138.95/2 to M.S., the UBS AG “im Auftrag eines Kunden,” and the Olga Mayenfisch Stiftung. P.G., P.H., U.H, and M.O.H. are supported by the Kanton of Zu¨rich. Received August 14, 2000; revised April 11, 2001. References

Detection of In Vivo Acetylation by Western Blot Analysis One ␮g of pmycFen1 was transfected into 293T cells. After 48 hr, nuclear extracts were prepared and myc-tagged Fen1 was immunoprecipitated using an anti-c-myc antibody (9E10). Immunocomplexes were washed three times with IP buffer with addition of 300 mM NaCl as described in Hasan et al. (2001). The proteins were resolved on SDS-PAGE and analyzed on Western blots for acetylation using an acetyl-group-specific antibody (PAN-AC1, abcam). The blots were reprobed using a Fen1-specific antibody. In Vitro Acetylation Assays Seven micrograms of purified Fen1 (or mutants thereof) were incubated with 0.1 ␮Ci [14C] acetyl coenzyme A (MC 269, Moravek Biochemicals) in 30 ␮l HAT-Buffer (50 mM Tris [pH 8.0], 10% [v/v] glycerol, 150 mM NaCl, 1 mM DTT, 1 mM PMSF, 10 mM Na butyrate) at 30⬚C for 30 min together with either immunoprecipitated p300 or glutathione sepharose bound GST-p300-HAT (100 ng). The reactions were subjected to SDS-PAGE analysis. Gels were stained with Coomassie and were subjected to autoradiography. Identification of Acetylated Lysine Residues Ten micrograms of bacterially purified Fen1 was incubated in the presence or absence of acetyl coenzyme A in 100 ␮l HAT-Buffer without PMSF and acetylated by glutathione sepharose bound GSTp300-HAT. Fifty microliters of the supernatant containing Fen1 was treated with 0.5 ␮g trypsin (Promega, Sequencing Grade, dissolved in 5 ␮l 0.1 M Tris [pH 8.2], 2mM CaCl2, 10% [v/v] acetonitrile) for 4 hr at 37⬚C. An aliqout of 10 ␮l was desalted on a ZipTip C18 (Millipore). Peptides were eluted with 1.5 ␮l of a saturated solution of ␣-cyano4-hydroxycinnamic acid in 0.1% (v/v) trifluoroacetic acid (TFA)/50% (v/v) acetonitrile and directly spotted onto the target of the MALDITOF mass spectrometer (Bruker, Biflex III). Acetylated peptides were identified by comparing the peptide masses determined for the

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