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J. Biol. Chem., Vol. 277, Issue 25, 22271-22278, June 21, 2002

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Direct Interaction between Uracil-DNA Glycosylase and a Proliferating Cell Nuclear Antigen Homolog in the Crenarchaeon Pyrobaculum aerophilum^*

Hanjing Yang, Ju-Huei Chiang, Sorel Fitz-Gibbon, Michel Lebel^§, Alessandro A. Sartori^¶, Joseph Jiricny^¶, Malgorzata M. Slupska, and Jeffrey H. Miller^**

From the Department of Microbiology and Molecular Genetics and the Molecular Biology Institute, University of California, Los Angeles, California 90095, ^§ Centre de Recherche en Cancerologie, Pavillon Hotel-Dieu de Quebec, CHUQ Quebec GIR 2J6, Canada, and ^¶ Institute of Medical Radiobiology of the University of Zürich and the Paul Scherrer institute, August Forel-Strasse 7, CH-8008 Zürich, Switzerland

Received for publication, February 22, 2002, and in revised form, March 27, 2002

	ABSTRACT

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Proliferating cell nuclear antigen (PCNA) acts as a sliding clamp on duplex DNA. Its homologs, present in Eukarya and Archaea, are part of protein complexes that are indispensable for DNA replication and DNA repair. In Eukarya, PCNA is known to interact with more than a dozen different proteins, including a human major nuclear uracil-DNA glycosylase (hUNG2) involved in immediate postreplicative repair. In Archaea, only three classes of PCNA-binding proteins have been reported previously: replication factor C (the PCNA clamp loader), family B DNA polymerase, and flap endonuclease. In this study, we report a direct interaction between a uracil-DNA glycosylase (Pa-UDGa) and a PCNA homolog (Pa-PCNA1), both from the hyperthermophilic crenarchaeon Pyrobaculum aerophilum (T_opt = 100 °C). We demonstrate that the Pa-UDGa-Pa-PCNA1 complex is thermostable, and two hydrophobic amino acid residues on Pa-UDGa (Phe¹⁹¹ and Leu¹⁹²) are shown to be crucial for this interaction. It is interesting to note that although Pa-UDGa has homologs throughout the Archaea and bacteria, it does not share significant sequence similarity with hUNG2. Nevertheless, our results raise the possibility that Pa-UDGa may be a functional analog of hUNG2 for PCNA-dependent postreplicative removal of misincorporated uracil.

	INTRODUCTION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Proliferating cell nuclear antigen (PCNA)¹ is essential for life. It is a processivity factor for DNA polymerase, forming a toroidal-shaped trimer acting as a sliding clamp on duplex DNA (1-4). Its function requires another protein, the clamp loader replication factor C, to load it onto the circular DNAs (5-9). PCNA is present in eukaryotes and its functional analog, the  subunit of DNA polymerase III holoenzyme, is present in bacteria (10, 11). More than a dozen classes of eukaryotic PCNA-binding proteins have been shown to interact with the PCNA sliding clamp, linking PCNA to several important biological processes beyond DNA replication, such as DNA repair and cell cycle regulation (12-18). In many cases, PCNA binding partners interact with PCNA through a conserved motif identified as QXX(L/M/I)XX(F/Y/H)(F/Y) that is usually located near either the amino or the carboxyl terminus. One important example of a eukaryotic PCNA-binding protein involved in DNA repair is human major nuclear uracil-DNA glycosylase (hUNG2), which removes uracil from misincorporated dUMP residues in an immediate postreplicative process (19, 20). hUNG2 interacts with PCNA through its PCNA binding site, ⁴QKTLYSFF¹¹, which is located near the amino terminus of hUNG2.

Recently PCNA sequence homologs have been identified in Archaea (21, 22). So far, each of the 10 completely sequenced archaeal genomes contains at least one putative PCNA homolog (23). There is a distinction found between the two major subdomains of the Archaea, Crenarchaeota and Euryarchaeota (23). Whereas each euryarchaeal genome tends to have one PCNA homolog, each crenarchaeal genome has two or three putative PCNA homologs (21, 23, 24). Biochemical studies have been conducted with several of the archaeal PCNA homologs, including a PCNA homolog from the euryarchaeote Pyrococcus furiosus and two PCNA homologs from the crenarchaeote Sulfolobus solfataricus (21, 22). These studies have confirmed that all of them are processivity factors for their corresponding DNA polymerases.

In Archaea, in addition to the PCNA clamp loader (replication factor C), two classes of archaeal proteins have so far been identified as PCNA-binding proteins, based on in vitro binding study and crystal structure analysis. They are family B DNA polymerase (Pol B) (22) and flap endonuclease (FEN) (25-29), both proteins known to interact with PCNA in eukaryotes. The proposed putative PCNA-binding motifs in these archaeal PCNA-binding proteins are quite similar to the conserved PCNA-binding motif identified in eukaryotic PCNA-binding proteins (22, 27). However, these putative PCNA binding sites have not been verified by mutation analysis.

In this study, we report identification of another archaeal PCNA-binding protein, Pyrobaculum aerophilum uracil-DNA glycosylase (Pa-UDGa), and the biochemical confirmation of its interaction with PCNA via the PCNA-binding motif. P. aerophilum is a hyperthermophile with an optimal growth temperature of 100 °C and a member of the crenarchaeal subdomain of Archaea (30). The biochemical characterization of uracil-DNA glycosylase activity of Pa-UDGa was recently published (31). Analysis of the complete genome sequence of P. aerophilum revealed two putative PCNA homologs (24), Pa-PCNA1 and Pa-PCNA2, as expected for a crenarchaeote (23). We demonstrate that Pa-UDGa preferentially binds to Pa-PCNA1, similar to two other P. aerophilum PCNA-binding proteins, Pa-FEN and P. aerophilum DNA polymerase B3 (Pa-Pol B3). Pa-UDGa's ability to bind to Pa-PCNA1 resembles the eukaryotic PCNA-binding protein hUNG2, which belongs to a distinctly different UDG family due to low amino acid sequence similarity to Pa-UDGa. Our results raise the possibility that Pa-UDGa may be a functional analog of hUNG2 for PCNA-dependent postreplicative removal of misincorporated uracil.

	EXPERIMENTAL PROCEDURES

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Bacterial Expression Plasmids-- P. aerophilum genomic DNA was prepared as described previously (32). The coding regions for Pa-UDGa (PAE0651, Protein Data Bank accession number AAL62921) and Pa-FEN (PAE0698, Protein Data Bank accession number AAL62961) were amplified by PCR using P. aerophilum genomic DNA as template with their corresponding primer pairs. The PCR products were cloned into a pCR2.1-TOPO vector using a TOPO TA cloning kit (Invitrogen). The primer information can be obtained upon request.

The full-length Pa-UDGa gene was amplified by PCR using pCR2.1TOPOPa-UDGa as template, cloned into a pGEX-2TK vector (Amersham Biosciences) at the BamHI site to create a plasmid that expresses a fusion protein of glutathione S-transferase (GST) and Pa-UDGa. The full-length Pa-FEN gene was amplified by PCR using pCR2.1TOPOPa-FEN as template and cloned into a pGEX-2TK vector (Amersham Biosciences) at the EcoRI site to create a plasmid that expresses GST-Pa-FEN fusion protein. Two Pa-Pol B3 (PAE2109, Protein Data Bank accession number AAL63952) fragments containing the carboxyl-terminal region (C1: amino acid residues 612-785, and C2: amino acid residues 726-785) were amplified by PCR using P. aerophilum genomic DNA as template, cloned into a pGEX-2TK vector (Amersham Biosciences) between the BamHI and EcoRI sites to create two plasmids that express GST-Pa-Pol B3 (C1: amino acids 612-785) fusion and GST-Pa-Pol B3 (C2: amino acids 726-785) fusion, respectively. The full-length Pa-PCNA1 gene (PAE3038, Protein Data Bank accession number AAL64629) was amplified by PCR using P. aerophilum genomic DNA, cloned into a pQE30 vector (Qiagen, Chatsworth, CA) between the BamHI and HindIII sites to create a plasmid that expresses the amino-terminal hexahistidine-tagged Pa-PCNA1. The full-length Pa-PCNA2 gene (PAE0720, Protein Data Bank accession number AAL62977) was amplified by PCR using P. aerophilum genomic DNA, cloned into a pQE30 vector (Qiagen) between the SphI and SalI sites to create a plasmid that expresses the amino-terminal hexahistidine-tagged Pa-PCNA2. The murine PCNA (33) was subcloned into a pBluescriptII KS vector (Stratagene, La Jolla, CA) between the BamHI and EcoRI sites. Subsequently, the BamHI-HindIII fragment was subcloned into a pQE30 vector (Qiagen) between the BamHI and HindIII sites to create a plasmid that expresses the amino-terminal hexahistidine-tagged murine PCNA.

For thermostable binding assays, the BamHI-BamHI fragment of Pa-UDGa was subcloned into pQE30 vector (Qiagen) to create a plasmid (pQE30Pa-UDGa) that expresses an amino-terminal hexahistidine-tagged Pa-UDGa recombinant protein. The BamHI-HindIII fragment of Pa-PCNA1 was subcloned into a pQE60 vector (Qiagen) to create a plasmid (pQE60Pa-PCNA1) that expresses the native form of Pa-PCNA1 without the histidine tag.

Generation of Pa-UDGa and Pa-FEN Mutants-- The amino acid fragments of Pa-UDGa 1-182, 131-196, and 172-196 were amplified by PCR using pCR2.1TOPOPa-UDGa as template. The products were subcloned into a pGEX-2TK vector (Amersham Biosciences) at the BamHI site to create GST fusion protein expression plasmids.

The Pa-UDGa mutant F183A/F184A was generated by standard site-directed mutagenesis procedure (34) using pCR2.1TOPOPa-UDGa as template. The obtained PCR products were cloned into a pGEX-2TK vector (Amersham Biosciences) at the BamHI site to create an expression plasmid for GST-Pa-UDGa (F183A/F184A), and the mutations were verified by DNA sequencing using a SequiTherm EXCEL II DNA sequencing kit (Epicentre, Madison, WI).

The Pa-UDGa mutant F191A/L192A was generated by site-directed mutagenesis. The PCR products were first cloned into a pCR2.1-TOPO vector using a TOPO TA cloning kit (Invitrogen). The BamHI-BamHI fragment containing the full-length Pa-UDGa with two amino acid substitutions was subcloned into a pGEX-2TK vector (Amersham Biosciences) at the BamHI site to create an expression plasmid for GST-Pa-UDGa (F191A/L192A), and the mutations were verified by DNA sequencing using a SequiTherm EXCEL II DNA sequencing kit (Epicentre).

The Pa-FEN mutant F345A/F346A was generated by site-directed mutagenesis. The PCR products were first cloned into a pCR2.1-TOPO vector using a TOPO TA cloning kit (Invitrogen). The EcoRI-EcoRI fragment containing the full-length Pa-FEN with two amino acid substitutions was subcloned into a pGEX-2TK vector (Amersham Biosciences) at the EcoRI site to create an expression plasmid for GST- Pa-FEN (F345A/F346A), and the mutations were verified by DNA sequencing using a SequiTherm EXCEL II DNA sequencing kit (Epicentre).

Expression and Partial Purification of Recombinant PCNA Homologs-- Overnight cultures of Escherichia coli BL21/pREP4 harboring plasmid pQE30 Pa-PCNA1, pQE30 Pa-PCNA2, or pQE30 murinePCNA were used to inoculate 100 ml of LB medium supplemented with 200 µg/ml ampicillin and 25 µg/ml kanamycin. The expression of Pa-PCNA1, Pa-PCNA2, or murine PCNA was induced with 0.3 mM isopropyl--D-thiogalactopyranoside during mid-log growth phase for 3 h at 37 °C. The plasmid pREP4 constitutively expresses the Lac repressor protein encoded by the lacI gene to reduce the basal level of expression (Qiagen). Cells were lysed in buffer A (20 mM Tris-HCl, pH 7.4, 60 mM NaCl, and 2 mM EDTA supplemented with 0.2 mg/ml lysozyme and 1 mM phenylmethylsulfonyl fluoride) by sonication. The cell lysates were clarified by centrifugation at 15,000 × g for 30 min. The protein concentration was determined by Bradford protein assay (Bio-Rad). The cell lysates were stored as small aliquots at 80 °C. Partial purification of Pa-PCNA1 and Pa-PCNA2 was obtained by heat treatment of 200 µl of cell lysates at 70 °C for 10 min followed by centrifugation at 15,000 × g for 30 min. The supernatant was stored at 80 °C.

A native Pa-PCNA1 recombinant protein without the histidine tag was obtained for the thermostable binding assay by a similar protocol as described above. An E. coli strain BL21/pREP4 harboring plasmid pQE60Pa-PCNA1 was used for the preparation of cell lysates. Subsequently, a heat treatment procedure was applied to the cell lysates to obtain a partial purified recombinant Pa-PCNA1 in its native form without the histidine tag.

Expression, Immobilization of GST Fusion Proteins, and "Pull-down" Affinity Bead Interaction Assay-- E. coli BL21 strain was used as the host to express all GST fusion proteins and the GST protein. The cell lysates were prepared in buffer A as described above. Protein concentration of the cell lysate was determined by Bradford protein assay (Bio-Rad). Cell lysates were incubated with the affinity matrices (GST-Sepharose beads, Amersham Biosciences) for 1 h at 4 °C. After extensive washing with buffer A, GST-Sepharose beads with immobilized GST fusion proteins were stored in buffer A at 4 °C. To determine the amount of each GST fusion protein bound for the pull-down assay, a fraction of GST-Sepharose bead-immobilized proteins was released by boiling in SDS sample buffer, analyzed by SDS-PAGE, and visualized by Coomassie Brilliant Blue staining.

The amount of each GST fusion protein bound used for the pull-down assay was adjusted for each sample based on the amount of bound proteins analyzed by SDS-PAGE. Extra GST-Sepharose beads were added to samples to bring the final amount of 60 µl. GST-Sepharose beads were mixed with PCNA samples at 4 °C for 1h. After washing six times with 0.8 ml of buffer A, the bound proteins were released by boiling in 60 µl of SDS sample buffer, analyzed by SDS-PAGE, and transferred to nitrocellulose membrane. Western blot analysis for Pa-PCNA homologs were performed using the manufacturer's protocols (Qiagen) with mouse anti-RGS(H)₄ primary antibody (1:500) and horseradish peroxidase-conjugated secondary antibody (1:5000) followed by ECL detection (Amersham Biosciences).

Thermostable Binding Assay-- An amino-terminal hexahistidine-tagged Pa-UDGa protein was expressed in an E. coli BL21/pREP4/pQE30Pa-UDGa strain, and the cell lysate was prepared by sonication in buffer B (50 mM sodium phosphate, pH 8.0, 300 mM NaCl, supplemented with 1 mM phenylmethylsulfonyl fluoride). To immobilize Pa-UDGa, Ni²⁺-NTA beads were added to the cell lysate and incubated at 4 °C for 1 h. After extensive washing with the buffer B, the Ni²⁺-NTA beads were transferred to buffer A. The Ni²⁺-NTA beads (60 µl) containing immobilized Pa-UDGa were mixed with the partial purified recombinant Pa-PCNA1 in its native form or with E. coli cell lysate containing pQE30 vector alone after 70 °C heat treatment. As a control for the experiment, a sample containing the partial purified Pa-PCNA1 and 60 µl of Ni²⁺-NTA beads without immobilized Pa-UDGa was also prepared. After washing five times with 0.8 ml of buffer A at 4 °C, the Ni²⁺-NTA beads were evenly split into two tubes. While one tube was incubated at 70 °C in a water bath for 5 min with 0.8 ml of buffer A pre-equilibrated at 70 °C, the other tube was kept on ice with 0.8 ml of chilled buffer A. Ni²⁺-NTA beads were pelleted at 1000 × g for 2 min. Bound proteins were released by boiling in SDS sample buffer, analyzed by SDS-PAGE, and visualized by Coomassie Brilliant Blue staining.

	RESULTS

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Expression and Partial Purification of Two Putative PCNA Homologs from P. aerophilum-- As a member of the archaeal subdomain Crenarchaeota, P. aerophilum contains two putative PCNA homologs, Pa-PCNA1 and Pa-PCNA2, identified by amino acid sequence homology (Fig. 1A; see also Ref. 24). Pa-PCNA1 shares 24% amino acid sequence identity with Pa-PCNA2 (Fig. 1B). The amino acid sequence identities between the two putative Pa-PCNA homologs and the other characterized eukaryotic or archaeal PCNAs range from 17 to 26% (Fig. 1B). Both putative Pa-PCNA homologs contain the highly conserved (L/I)AP(K/R) motif located near the carboxyl terminus, which may interact with the clamp loader, replication factor C (23). Phylogenetic analysis of PCNA homologs in Crenarchaea reveals that the multiple homologs in Crenarchaea fall into two classes, consistent with a duplication event early after the divergence of the crenarchaeal clade (23).

View larger version (103K): [in this window] [in a new window]   Fig. 1.   Sequence alignment of Pa-PCNA1, Pa-PCNA2, and characterized eukaryotic and archaeal PCNA homologs using the program ClustalW (41) (A) and percentage of sequence identity among PCNA homologs shown in panel A (B). The homologs are the following (organisms and the Protein Data Bank accession numbers in parentheses): Hsa-PCNA (Homo sapiens, P12004); Spo-PCNA (Schizosaccharomyces pombe, CAB38513); Pfu-PCNA (P. furiosus, BAA33020); Pa-PCNA1 (P. aerophilum PCNA1, AAL64629); Sso-PCNA A (039p) (S. solfataricus, P57765); Pa-PCNA2 (P. aerophilum, AAL62977); Sso-PCNA B (048p) (S. solfataricus, P57766). Black boxes indicate 70-100% amino acid residue identity, and gray boxes indicate 70-100% similarity. The asterisks mark amino acid residues corresponding to those forming the hydrophobic pocket in human PCNA (17, 23).

Pa-PCNA1 and Pa-PCNA2 were cloned on the bacterial expression vector pQE30 and expressed as amino-terminal hexahistidine-tagged recombinant proteins in E. coli (Fig. 2, A and B, lanes 3 and 5). To obtain partially purified recombinant Pa-PCNA homologs for in vitro binding assays, we took advantage of the heat stability characteristic of proteins from thermophiles and heated the E. coli crude cell lysates expressing each homolog to 70 °C. The recombinant Pa-PCNA1 and Pa-PCNA2 were largely heat-stable after a 10-min heat treatment (Fig. 2, A and B, lanes 4 and 6). A hexahistidine-tagged eukaryotic murine PCNA recombinant protein was also included in the experiment and was found to be heat-labile under the conditions tested (Fig. 2, A and B, lanes 7 and 8). Although the recombinant Pa-PCNA1 and Pa-PCNA2 have predicted molecular masses of 28 and 29 kDa, respectively, the apparent molecular mass on the SDS-polyacrylamide gel was ~37 kDa for Pa-PCNA1 and 32 kDa for Pa-PCNA2. The aberrant migration of Pa-PCNAs on the SDS-polyacrylamide gel was also seen in two S. solfataricus PCNAs (21) and eukaryotic PCNA homologs (Fig. 2A, lane 7; see also Ref. 35) for unknown reasons.

View larger version (93K): [in this window] [in a new window]   Fig. 2.   Partial purification of recombinant Pa-PCNA1 and Pa-PCNA2. The arrows mark full-length recombinant proteins. A, SDS-PAGE analysis of crude E. coli lysates containing hexahistidine-tagged Pa-PCNA1 and hexahistidine-tagged Pa-PCNA2 before the heat treatment (4 °C) and after the heat treatment (70 °C). Vector, pQE30 without insert (lanes 1 and 2); Pa-PCNA1, pQE30Pa-PCNA1 (lanes 3 and 4); Pa-PCNA2, pQE30Pa-PCNA2 (lanes 5 and 6); and mPCNA, pQE30murinePCNA (expresses hexahistidine-tagged murine PCNA, lanes 7 and 8). The crude E. coli lysates were prepared as described under "Experimental Procedures," subjected to SDS-PAGE, and visualized by Coomassie Blue staining. B, Western blot analysis of the SDS gel shown in panel A. The Western blotting procedure was performed as described under "Experimental Procedures."

In Vitro Direct Binding of Pa-Pol B3 to Pa-PCNA1-- Pa-Pol B3 was tested for binding to recombinant Pa-PCNA1 and Pa-PCNA2. Analysis of the Pa-Pol B3 protein sequence revealed that its carboxyl-terminal region contains a putative PCNA-binding motif ⁷⁷⁸ERTLLDFF⁷⁸⁵ (Fig. 3A), which is similar to the putative PCNA-binding motifs of several archaeal Pol B homologs predicted by Ishino and colleagues (22). Glutathione S-transferase (GST) fusion proteins were constructed for two overlapping fragments containing the carboxyl-terminal region of Pa-Pol B3 (C1: amino acids 612-785, C2: amino acids 726-785, Fig. 3, A and B). In the pull-down affinity bead interaction assays, GST alone had no detectable binding to either of the Pa-PCNAs (Fig. 3C, lanes 2 and 6). However, binding to Pa-PCNA1 was detected with both of the GST-Pa-Pol B3 fusions (Fig. 3C, lanes 3 and 4). In addition, a weak binding to Pa-PCNA2 was also detected (Fig. 3C, lanes 7 and 8). The observed preferred binding to Pa-PCNA1 by the carboxyl-terminal region of Pa-Pol B3 provides evidence for an in vivo direct interaction between Pa-Pol B3 and Pa-PCNA1.

View larger version (38K): [in this window] [in a new window]   Fig. 3.   In vitro direct binding between Pa-PCNA1 and the carboxyl-terminal region of Pa-Pol B3. A, schematic diagram of Pa-Pol B3 and the two fragments (C1 and C2). B, SDS-PAGE analysis of the following samples: GST (lane 2), GST-Pol B3 (C1, amino acids 612-785) (lane 3), and GST-Pol B3 (C2, amino acids 726-785) (lane 4). Lane M contains molecular mass standards (Bio-Rad) as indicated on the left. The protein bands were visualized by Coomassie Blue staining. C, Western blot analysis of the samples from the pull-down experiment: inputs of Pa-PCNA1 and Pa-PCNA2 (lanes 1 and 5); GST alone with Pa-PCNA1 and Pa-PCNA2 (lanes 2 and 6); GST-Pol B3 (C1) with Pa-PCNA1 and Pa-PCNA2 (lanes 3 and 7); GST-Pol B3 (C2) with Pa-PCNA1 and Pa-PCNA2 (lanes 4 and 8). Sample preparation and Western blot analysis are described under "Experimental Procedures."

In Vitro Direct Binding of Pa-UDGa and Pa-FEN to Pa-PCNA1-- A GST fusion protein with Pa-UDGa was constructed for the study of in vitro direct binding of Pa-UDGa to two recombinant Pa-PCNA homologs using the pull-down affinity bead interaction assay (Fig. 4A, lane 3). Pa-FEN, which was predicted to be a PCNA-binding protein based on previous studies (27), was also included for the binding experiment (Fig. 4A, lane 4). Although GST alone bound to neither Pa-PCNA (Fig. 4B, lanes 3 and 4), both GST-Pa-UDGa and GST-Pa-FEN bound to Pa-PCNA1 (Fig. 4B, lanes 5 and 7). Binding to Pa-PCNA2 was not detected with either GST fusion (Fig. 4B, lanes 6 and 8). Therefore, the observed binding of Pa-UDGa and Pa-FEN to Pa-PCNA1 provides evidence for a direct in vivo interaction between Pa-UDGa-Pa-PCNA1 and Pa-FEN-Pa-PCNA1. The effect of NaCl concentration on the formation of a complex between Pa-UDGa and Pa-PCNA1 was studied. Binding was detected in the presence of 0.05-0.4 M NaCl and was disrupted at NaCl concentrations higher than 0.4 M (Fig. 4, C and D). The effect of temperature on the formation of the complex was also studied. When using a recombinant hexahistidine-tagged Pa-UDGa and a recombinant Pa-PCNA1 in its native form without any tag, binding was observed at 4 °C and was largely retained after a 5-min treatment at 70 °C (Fig. 4E, lanes 3 and 4). Under the same experimental conditions, the recombinant Pa-PCNA1 protein alone had no detectable binding to the Ni²⁺-NTA resin (data not shown). Binding of Pa-UDGa and Pa-FEN to the eukaryotic murine PCNA was also tested but was undetectable under our experimental conditions (data not shown).

View larger version (42K): [in this window] [in a new window]   Fig. 4.   In vitro direct binding of Pa-UDGa and Pa-FEN to Pa-PCNA1, the effect of NaCl concentration and temperature. A, SDS-PAGE analysis of the following samples: GST (lane 2), GST-Pa-UDGa (lane 3), and GST-Pa-FEN (lane 4). Lane M contains molecular mass standards (Bio-Rad) as indicated on the left. The protein bands were visualized by Coomassie Blue staining. B, Western blot analysis of the samples from the pull-down experiment: inputs of Pa-PCNA1 and Pa-PCNA2 (lanes 1 and 2); GST alone with Pa-PCNA1 and Pa-PCNA2 (lanes 3 and 4); GST-Pa-UDGa with Pa-PCNA1 and Pa-PCNA2 (lanes 5 and 6); GST-Pa-FEN with Pa-PCNA1 and Pa-PCNA2 (lanes 7 and 8). C, Western blot analysis of the samples from the pull-down experiment of GST-Pa-UDGa with Pa-PCNA1 in the presence of varying amounts of NaCl as indicated. The position of Pa-PCNA1 was marked on the right. D, SDS-PAGE analysis of the amount of full-length GST-Pa-UDG fusion protein used in the pull-down experiment shown in panel C. The protein bands were visualized by Coomassie blue staining. The position of the full-length GST-Pa-UDGa was marked on the right. E, thermostable binding between Pa-UDGa and Pa-PCNA1. SDS-PAGE of the following samples from the pull-down experiment: input of Pa-PCNA1 without any tag (lane 2); His6-tagged Pa-UDGa with Pa-PCNA1 without 70 °C treatment (lane 3) and with 70 °C treatment (lane 4). The protein bands were visualized by Coomassie blue staining. Lane M contains molecular mass standards (Bio-Rad) as indicated on the left. Sample preparation and Western blot analysis are described under "Experimental Procedures."

The PCNA Interaction Motif Is Located near the Carboxyl Terminus of Pa-UDGa-- The interaction between Pa-UDGa and Pa-PCNA1 was further verified by identifying the specific regions on Pa-UDGa required for PCNA binding. First, three GST fusion proteins that contain various regions of Pa-UDGa were constructed and tested for Pa-PCNA1 binding activity using the pull-down affinity bead interaction assay (Fig. 5A). GST-Pa-UDGa (amino acids 1-182) did not bind to Pa-PCNA1, and this fusion protein had an excessive proteolytic degradation (data not shown). However, both GST fusion proteins containing the carboxyl-terminal region of Pa-UDGa displayed binding activity (data not shown for GST-Pa-UDGa (amino acids 131-196); see Fig. 5, C and D, lane 8 for GST-Pa-UDGa (amino acids 172-196)). These results demonstrate that the 25-amino acid region near the Pa-PCNA1 carboxyl terminus is required for PCNA binding.

View larger version (33K): [in this window] [in a new window]   Fig. 5.   PCNA binding activity of the carboxyl-terminal region of Pa-UDGa. A, schematic diagram summarizing the results of the PCNA binding assays with GST fusions containing different Pa-UDGa amino acid segments as indicated. B, schematic diagrams of the Pa-UDGa and Pa-FEN proteins. The putative PCNA-binding motifs located at the carboxyl-terminal region of Pa-UDGa and Pa-FEN are indicated. C and D, SDS-PAGE analysis (C) and Western blot analysis (D) of the following samples from the pull-down experiment: input of Pa-PCNA1 (lane 1); GST alone with Pa-PCNA1 (lane 2); GST-wild type Pa-FEN (WT, lane 3) and GST-mutant Pa-FEN F345A/F346A (FFAA, lane 4) with Pa-PCNA1; GST-wild-type Pa-UDGa (WT, lane 5) and three GST-Pa-UDGa mutants with Pa-PCNA1: F183A/F184A (FFAA, lane 6); F191A/L192A (FLAA, lane 7), and carboxyl-terminal (amino acids 172-196, lane 8). The position of Pa-PCNA1 was marked on the right. The molecular mass standards (Bio-Rad) are indicated on the left. Sample preparation and Western blot analysis are described under "Experimental Procedures."

Next, Pa-UDGa mutants with specific substitutions within the above identified 25-amino acid region were generated to identify amino acid residues critical for PCNA binding activity. Previous studies have shown that many PCNA-binding proteins contain the consensus PCNA-binding motif QXX(L/M/I)XX(F/Y/H)(F/Y), and mutational analysis indicates that the two consecutive hydrophobic amino acid residues within this motif are involved in the interaction with PCNA (36). Analysis of the Pa-UDGa amino acid sequence revealed two putative PCNA-binding motifs near the carboxyl terminus of Pa-UDGa (Fig. 5B). The sequence for the first region is ¹⁷⁷QKDLAMFF¹⁸⁴ (motif 1), which contains the eukaryotic PCNA-binding consensus sequence QXXLXXFF. The sequence for the second region is ¹⁸⁵GGGLDRFL¹⁹² (motif 2), which contains two consecutive hydrophobic amino acid residues (Phe¹⁹¹ and Leu¹⁹²) closer to the carboxyl terminus (Fig. 5B). Two Pa-UDGa mutants were generated for the pull-down binding assay, each with two amino acid changes in the putative PCNA-binding motif 1 (F183A/F184A) or motif 2 (F191A/L192A). A Pa-FEN mutant carrying the F345A/F346A modification in its putative PCNA-binding motif ³³⁹TSSLDSFF³⁴⁶ near its carboxyl terminus was also included in the experiment (Fig. 5B). As expected, the Pa-FEN mutant carrying F345A/F346A failed to bind to Pa-PCNA1 (Fig. 5, C and D, lane 4). The Pa-UDGa mutant carrying F183A/F184A in the putative binding motif 1 was still capable of binding to Pa-PCNA1 (Fig. 5, C and D, lane 6). However, the binding was largely abolished in the Pa-UDGa mutant carrying F191A/L192A in the putative motif 2 (Fig. 5, C and D, lane 7). These results show that Phe³⁴⁵ and Phe³⁴⁶ of Pa-FEN, and Phe191 and Leu¹⁹² of Pa-UDGa are necessary for the binding of Pa-FEN and Pa-UDGa to Pa-PCNA1.

	DISCUSSION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

With the goal of finding new archaeal PCNA-binding proteins, we searched the predicted protein sequences of the P. aerophilum genome with identified putative PCNA-binding motifs using MACPATTERN 3.6 (37). In this way, Pa-UDGa was identified as a putative PCNA-binding protein. We carried out in vitro biochemical analysis of Pa-UDGa binding to two putative P. aerophilum PCNA homologs, Pa-PCNA1 and Pa-PCNA2, using the pull-down affinity bead interaction assay. Our results show that Pa-UDGa preferentially binds to Pa-PCNA1 to form a thermostable protein complex. Apparently, the binding between Pa-UDGa and Pa-PCNA1 is specific as Pa-UDGa has no detectable binding to the murine PCNA. Two consecutive hydrophobic amino acid residues, Phe¹⁹¹ and Leu¹⁹², located near the carboxyl terminus of Pa-UDGa, are crucial for PCNA binding activity. Maintaining these same experimental conditions, the interactions between Pa-PCNA1 and two other P. aerophilum proteins, Pa-FEN (wild type and mutant F345A/F346A) and the carboxyl-terminal region of Pa-Pol B3, provides evidence for Pa-PCNA1 as a functional PCNA homolog in P. aerophilum. At this point, we were unable to perform in vivo experiments to verify the physiological significance of the PCNA binding property of Pa-UDGa due to the current lack of a genetic system in P. aerophilum. However, identification of Pa-UDGa as an archaeal PCNA-binding protein and its critical amino acid residues for PCNA binding is the first step in revealing its biological significance in future genetic analyses.

The PCNA binding activity observed in Pa-UDGa from this study and in hUNG2 from previous studies (19) raises the possibility that Pa-UDGa may be a functional analog of hUNG2. Pa-UDGa and hUNG2 belong to two distinct UDG families due to the low sequence similarity between them (31, 38). In addition, Pa-UDGa is an olive green-colored protein (31) due to the presence of an iron-sulfur cluster (39), whereas hUNG2 is largely colorless. Despite these differences, both Pa-UDGa and hUNG2 have similar uracil glycosylase activities, and both interact with PCNA through specific binding domains at either the carboxyl-terminal region (for Pa-UDGa) or the amino-terminal region (for hUNG2, 19). Thus, it is possible that Pa-UDGa may be a functional analog of hUNG2 for PCNA-dependent postreplicative removal of misincorporated uracil (19, 20).

At this point, two or three putative PCNA homologs have been identified in the completely sequenced Crenarchaeota genomes, including P. aerophilum, S. solfataricus, and Aeropyrum pernix (21, 23). The presence of more than one PCNA homolog may reflect either functional redundancy or functional differentiation. Previous experiments with S. solfataricus SsoPCNA A (039p) and SsoPCNA B (048p) demonstrate that both SsoPCNA homologs are processivity factors for the single-subunit family B DNA polymerase (Pol B1) despite their slightly different efficiencies (21). Our study with Pa-PCNA1 and Pa-PCNA2 suggests a possible functional differentiation between two Pa-PCNA homologs. Binding to Pa-UDGa and Pa-FEN is only detected with Pa-PCNA1. Similarly, the carboxyl-terminal region of Pa-Pol B3 also preferentially binds to Pa-PCNA1. Although we cannot eliminate the possibility that the recombinant Pa-PCNA2 is somehow inactivated under our experimental conditions, regardless of its heat stability and weak binding to the carboxyl-terminal region of Pa-Pol B3, our results suggest that Pa-PCNA1 may be the major protein with functions similar to PCNA in eukaryotes. Whether Pa-PCNA2 is functional, and if so, which proteins it might interact with, remains to be answered. Our experiments do not rule out the possibility that Pa-PCNA1 and Pa-PCNA2 monomers may combine to make functional heteromeric trimers.

Although FEN homologs are present in Archaea and Eukarya (27), Pa-UDGa family members are found in Archaea and Eubacteria (38, 40). The amino acid sequence alignments of the carboxyl-terminal regions of FEN and UDG homologs are shown in Fig. 6. Although the conserved PCNA-binding motif QXXLXX(F/W)F was observed near the carboxyl terminus for almost all archaeal FEN homologs (Fig. 6A; see also Ref. 27), divergent carboxyl-terminal sequences were observed for archaeal UDG homologs (Fig. 6B). Only four homologs contained recognizable PCNA-binding motifs: Ape-UDG from A. pernix, Sso-UDG1 from S. solfataricus, Af-UDG from Archaeoglobus fulgidus, and Halo-UDG from Halobacterium sp. NRC-1. Biochemical experiments will be required to test these binding motifs and to determine whether the remaining archaeal homologs also bind PCNA.

View larger version (52K): [in this window] [in a new window]   Fig. 6.   Sequence alignment of the carboxyl-terminal regions of archaeal FEN homologs and archaeal UDG homologs using the ClustalW program (41). Black boxes indicate 70-100% amino acid identity, and gray boxes indicate 70-100% amino acid similarity. A, carboxyl-terminal region of FEN. The asterisks mark the proposed PCNA-binding motif (27). The symbol () marks a eukaryotic FEN homolog. The homologs are the following (organisms and the Protein Data Bank accession numbers in parentheses): Tv-FEN (Thermoplasma volcanium, BAB59701; Ta-FEN (Thermoplasma acidophilum, CAC12164); Ape-FEN (A. pernix, BAA79026); Sso-FEN (S. solfataricus, AAK40525); Pa-FEN (P. aerophilum, AAL62961); Af-FEN (A. fulgidus, AAB90967); Pab-FEN (Pyrococcus abyssi, CAB49654); Ph-FEN (Pyrococcus horikoshii, BAA30521); Mj-FEN (Methanococcus jannaschii, AAB99454); Mth-FEN (Methanobacterium thermoautotrophicum, AAB86106); Halo-FEN (Halobacterium sp. NRC-1, AAG19690); and Hsa-FEN (Homo sapiens, AAH00323). B, carboxyl-terminal region of UDG. The boxed amino acid residues are proposed to be involved in PCNA interaction. The symbol () indicates a eubacterial UDG homolog. The homologs are the following (organisms and the Protein Data Bank accession numbers in parentheses): Pa-UDGa (P. aerophilum, AAL62921); Ape-UDG (A. pernix, BAA79385); Sso-UDG1 (S. solfataricus, AAK42437); Af-UDG (A. fulgidus, AAB88977); Pab-UDG (P. abyssi, CAB49606); Ph-UDG (P. horikoshii, BAA30579); Aa-UDG (Aquifex aeolicus, AAC07559); Tm-UDG (Thermotoga maritima, AAD35596); Tp-UDG (Treponema pallidum, AAC65215); Halo-UDG (Halobacterium sp. NRC-1, AAG20230); Ta-UDG (T. acidophilum, CAC11619); and Tv-UDG (T. volcanium, BAB59983).

	FOOTNOTES

^* This work was supported by National Research Service Award No. T32 CA09056 from the United States Health and Human Services Institutional (to H. Y.), by Grant GM 57917 from the National Institutes of Health (to J. H. M.) and by the generous financial support of the Union Bank of Switzerland Stiftung (to A. A. S).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Present address: Center for Astrobiology, Institute of Geophysics and Planetary Physics, UCLA, Los Angeles, CA 90095.

Present address: Diversa, 4995 Directors Place, San Diego, CA 92121

^** To whom correspondence should be addressed: Dept. of Microbiology and Molecular Genetics, 1602 Molecular Sciences Bldg., 405 Hilgard Ave., Los Angeles, CA 90095. Tel.: 310-825-8460; Fax: 310-206-3088; E-mail: jhmiller@mbi.ucla.edu.

Published, JBC Papers in Press, April 1, 2002, DOI 10.1074/jbc.M201820200

	ABBREVIATIONS

The abbreviations used are: PCNA, proliferating cell nuclear antigen; UDG, uracil-DNA glycosylase; Pa-UDGa, P. aerophilum UDGa; Pol B, family B DNA polymerase; Pa-Pol B3, P. aerophilum Pol B3; FEN, flap endonuclease; GST, glutathione S-transferase; Ni²⁺-NTA, nickel-nitrilotriacetic acid; Pa, P. aerophilum; hUNG2, human major nuclear uracil-DNA glycosylase.

	REFERENCES

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

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