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PCNA binding proteins in Drosophila melanogaster : the analysis of a conserved PCNA binding domain
Introduction
Materials And Methods
Plasmid expression constructs and yeast two-hybrid methods
Peptide analysis
ELISA analysis of PCNA binding to peptides
Competition ELISA
Peptide pull-down experiments
Sequence analysis and profile searching
Results
Two-hybrid screening identifies PCNA-binding proteins from Drosophila
Pogo and Dacapo proteins bind to the same region of PCNA
Pogo and Dacapo proteins contain conserved consensus PCNA-binding domains
Use of the PCNA consensus as a tool for predicting PCNA-binding proteins
Discussion
Dacapo is a member of the p21WAF1/Cip1 family of CDK inhibitors
The Pogo transposase interacts with PCNA
Molecular analysis of the PCNA interaction
Summary
Acknowledgements
References
PCNA binding proteins in Drosophila melanogaster : the analysis of a conserved PCNA binding domain
ABSTRACT
INTRODUCTION
Proliferating cell nuclear antigen (PCNA) plays an essential role in both the replication and repair of DNA. It forms a toroidal shaped trimer which interacts non-specifically with DNA by encircling it, forming a sliding clamp which tethers the polymerase complex to the DNA duplex. PCNA is highly conserved and has been identified in a very wide range of eukaryotes (1,2). Genes showing sequence homology with PCNA have been identified as far down the evolutionary ladder as the Archaea, whose DNA replication and repair machinery appears to be more typically eukaryotic then prokaryotic (3). The [beta] subunit of Escherichia coli polymerase III is a functional prokaryotic homologue of PCNA and although this protein shows six-fold symmetry like PCNA, it exists as a dimer rather than a trimer (4).
PCNA is an essential component of the DNA replication machinery, acting as the processivity factor for polymerases [delta] and [epsis] (5,6). In addition to its role in replication, PCNA is also required for nucleotide excision repair (7,8) and plays a role in one pathway of base excision repair (9). Recent analysis of proteins which interact with PCNA have shown that it interacts not only with enzymes involved in the mechanics of DNA repair and replication, but also binds to cell cycle regulatory proteins such as p21 and Gadd45 (10-14). This suggests that the interaction of PCNA with other proteins is a key regulatory target for the co-ordination of DNA replication and repair.
PCNA in Drosophila is encoded by the mus209 gene: mutations in mus209 result in lethality, although several mus209 mutants are temperature sensitive and provide unique tools to analyse the functions of PCNA in a developing multicellular organism (15). PCNA is required throughout development and maternally encoded PCNA is essential for embryogenesis (15). Analysis of the 5[prime]-flanking region of mus209 indicates that its transcription is regulated by homeodomain proteins, suggesting that its expression is coordinated with cell proliferation and differentiation (16). The mus209 mutants are highly sensitive to a range of DNA damaging agents, supporting the role of PCNA in DNA repair. The suppression of position effect variation seen in these mutants points to a role for PCNA in chromatin assembly (15).
In a screen for proteins which interact with Drosophila PCNA, we have identified several interacting proteins. Two of them, which we describe here, have not previously been identified as PCNA-binding proteins. One is the Dacapo protein, which is a cyclin-dependent kinase inhibitor (17,18). The second is the transposase encoded by the Pogo transposon, which belongs to the TC1/Mariner superfamily of transposons (19). We find that both proteins contain a consensus PCNA-binding domain shared by other PCNA-binding proteins such as p21, Fen1, XPG and MCMT (12,20-23). We find that a human relative of Pogo, named Tigger, also potentially encodes a protein containing a conserved PCNA-binding domain (24). This raises interesting possibilities for a novel method of tranposition of such elements, possibly by targeting the transposase to replicating DNA.
MATERIALS AND METHODS
Plasmid expression constructs and yeast two-hybrid methods
Manipulations of E.coli and DNA were by standard methods (25). Double-stranded plasmid DNA was sequenced using the Sequenase[trade] protocol (US Biochemical). The plasmids expressing human, Drosophila and Schizosaccharomyces pombe PCNA have been described (12,14). Growth and maintenance of Saccharomyces cerevisiae was according to standard methods (26). Transformation was carried out by the method of Gietz et al. (27). The S.cerevisiae strain Y190 (MATa leu2-3,112 ura3-52 trp1-901 his3-D200 ade2-101 gal4D gal80D cyhR URA3::GAL1-lacZ LYS2::GAL1-HIS3) was used for all two-hybrid analysis, which expresses the reporter genes lacZ (E.coli) and HIS3 (S.cerevisiae) under the control of the GAL1 promoter. All two-hybrid screening and analysis was carried out as previously described (12,20). pACT plasmids identified in the two-hybrid screening experiment were tested against pAS plasmids encoding unrelated Gal4 fusion proteins to exclude false positive results: these included pAS-Snf1, pAS-p53, pAS-Cdk2 and pAS-lamin (20).
Peptide analysis
The following 20 amino acid peptides were used (Chiron Mimotopes, Australia). These peptides were linked via residues SGSG at the N-terminus to biotin. p21, KRRQTSMTDFYHSKRRLIFS; p21-A, KRRATSMTDFYHSKRRLIFS; Dacapo, RKRQPKITEFMKERKRLAQA; Dacapo-A, RKRAPKITEFMKERKRLAQA; Z50796, TKRQQKMTDFMAVSRKKNSL; Z50796-A, TKRQQKMTDFMAVSRKKNSL; Pogo, KLFNLHINSAVLQKKITDYF; Pogo-A, KLFNLHINSAVLAKKITDYF; Tigger, LMWQTSLLSYFKKLPQPPQP; Tigger-A, LMWQTSLLSYFKKLPQPPQP; Consensus, KKRQKRLTDFFKRKRKLKEA; Consensus-A, KKRAKRLTDFFKRKRKLKEA; Unrelated, PESVELKWSEPNEEELIKFM.
ELISA analysis of PCNA binding to peptides
Plastic plates for enzyme-linked immunoabsorption assay (ELISA) were coated with 100 µl 5 µg/ml streptavidin by drying overnight at 37°C. They were washed with phosphate-buffered saline (PBS) containing 0.2% Tween 20 (PBST) and blocked with 5% non-fat milk powder in PBS for at least 2 h at room temperature. Each well was incubated in turn with the following reagents (the wells were extensively washed in PBST between steps): (i) 0.5 µg peptide diluted in 100 µl 0.1% milk-PBS (2 h, room temperature); (ii) up to 50 µg protein in 100 µl 0.1% milk-PBS, either total cell lysates of E.coli BL21 expressing human or S.pombe PCNA or purified human or S.pombe PCNA (1 h, room temperature); (iii) primary anti-PCNA rabbit polyclonal antiserum 3009 or PC10 monoclonal anti-PCNA antibody diluted 1 in 2000 in 2% milk-PBS (1 h, room temperature); (iv) secondary horseradish peroxidase (HRP)-conjugated anti-rabbit or anti-mouse antibodies diluted 1 in 1000 in 2% milk-PBS (1 h, room temperature); (v) 50 µl chromogenic substrate TMB at 100 µg/ml in 0.1 M sodium acetate, pH 6.0, containing a 1 in 1000 diluted solution of H2O2 (30%). Once the colour reaction had developed, the reaction was stopped by adding 50 µl 1 M H2SO4 and the plate read using a Dynatech 5000 ELISA plate reader at 450 nm.
Competition ELISA
Peptide ELISA experiments were carried out as described above, with competing peptides added to the PCNA solution before addition to the immobilized, biotinylated peptides. A p21-derived peptide (KRRQTSMTDFYHSKRRLIFS) or an unrelated control peptide (KPVRLPSIQAIPCAP) were dissolved in DMSO at 20 µg/mg and diluted in DMSO to various concentrations. The DMSO-diluted peptides were then diluted 1 in 200 into a solution of 0.1% milk-PBST containing purified human PCNA at 5 µg/ml, immediately before adding to the peptide-coated wells.
Peptide pull-down experiments
An aliquot of 2.7 µg each peptide was incubated with 10 µl streptavidin-agarose beads (Sigma) in PBS for 1 h at room temperature, then the beads were washed extensively in PBS and recovered each time by centrifugation. A sample of 20 µl Drosophila embryonic extract was added to the washed beads at a final concentration of 1 mg/ml and incubated with the beads on ice for 1 h. The beads were extensively washed in PBS containing 0.05% Tween 20 and bound proteins removed by boiling in SDS loading buffer. Proteins were separated by 15% SDS-PAGE then electrophoretically transferred to PVDF membrane (Amersham). The membranes were blocked in PBS containing 2% skimmed milk for 30 min, then incubated for 1 h with a polyclonal rabbit anti-Drosophila PCNA antibody diluted 1 in 1000 in 2% skimmed milk-PBS. After washing, blots were incubated with secondary HRP-conjugated swine anti-rabbit antibodies diluted 1 in 1000 in 2% skimmed milk-PBS for 30 min, followed by washing in PBS containing 0.05% Tween 20. Bound antibody was visualized using the ECL system according to the manufacturer's instructions (Amersham).
Sequence analysis and profile searching
Sequence analysis was carried out using the UWGCG programs at the Daresbury Seqnet facility and BLAST programs at the NCBI web site (27,28). For profile searching, various combinations of the amino acid sequences shown in Figure
RESULTS
Two-hybrid screening identifies PCNA-binding proteins from Drosophila
In order to identify Drosophila proteins which interacted with PCNA, we employed a yeast-based two-hybrid interaction screening approach. Full-length Drosophila PCNA was expressed in yeast as a fusion protein with the DNA-binding domain of S.cerevisiae Gal4 (12). This strain was then transformed with a cDNA library in pACT derived from third instar larvae of Drosophila melanogaster which expresses hybrid fusion constructs with the transcriptional activation domain of Gal4 (30). Transformants where the two forms of hybrid protein interacted were detected by their ability to form functionally active Gal4 complexes and thus activate Gal4-dependent reporter constructs. Approximately 5 × 106 transformants were screened for their ability to grow on medium containing 3-aminotriazole as a test for expression of the reporter gene HIS3+. Of 55 His+ colonies, 20 also expressed detectable levels of [beta]-galactosidase, indicating that the other reporter gene, lacZ, was also expressed. The pACT plasmids from these transformants were isolated and tested against plasmids expressing various unrelated protein fusions to test for specificity of interaction (Materials and Methods). From sequence analysis, the clones fell into several classes, two of which are described here.
Pogo and Dacapo proteins bind to the same region of PCNA
Figure 1. Schematic diagram of the protein sequences encoded by the Dacapo clones identified in the two-hybrid screen against Drosophila PCNA. The red box (residues 31-111) represents the consensus cyclin/CDK-binding domain. The blue box indicates the position of the PCNA-binding consensus (residues 118-197) and these residues are shown beneath, along with the conserved domain from C.elegans, mouse and human, where the blue boxes indicate highly conserved residues within the domain. Three overlapping clones encoded the Dacapo protein, which has been identified as encoding a cyclin-dependent kinase inhibitor belonging to the p21/p27 class (17,18; Fig. Three clones were identified in the two-hybrid screen which contained sequences identical to the Pogo transposon (19; Fig. Figure 2. (Top) A schematic diagram of the Pogo transposon, showing TIRs as black boxes and the two large ORFs which encode the Pogo transposase. The regions corresponding to the inserts identified in the two-hybrid screen against Drosophila PCNA are shown. The clones pDMpip11, pDMpip9 and pDMpip12 contained cDNA inserts of ~1.8, 1.6 and 0.3 kb, respectively. (Bottom) An alignment between the C-terminal sequences of Pogo and the second ORF of Tigger2. Identical residues are shown and + indicates a conserved change. Since PCNA is a highly conserved eukaryotic protein, we used the two-hybrid system to determine whether Dacapo and Pogo could bind to PCNA from human and S.pombe. We found that in both cases the interaction was evolutionarily conserved (data not shown). Since these proteins interact with human PCNA, it was possible to test them using the yeast two-hybrid interaction assay against a panel of human PCNA subclones designed for analysing the domain specificity of human PCNA-binding proteins. The results of these experiments show that Pogo and Dacapo bind to the same region of PCNA as p21 and Fen1, which includes the interdomain linker region (12,20; Fig. Figure 3. Two-hybrid domain analysis of PCNA binding to Dacapo and Pogo. The PCNA monomer showing the two structural domains is represented schematically. A series of N- and C-terminal deletions of human PCNA (12) were expressed as fusions with the DNA-binding domains of Gal4. These were tested for their interactions with the Dacapo- and Pogo-encoding two-hybrid isolates shown in Figures 1 and 2 as described in Material and Methods: each clone showed the same pattern of interaction. These results were compared with those of previous experiments testing the pattern of interaction of Fen1, p21 and Gadd45 with these PCNA constructs (12,14,20). These results suggest that p21, Fen1, Dacapo and Pogo interact with the same region of PCNA, while Gadd45 shows a different pattern of binding. Examination of the smallest clones of Dacapo and Pogo identified by two-hybrid screening (Figs In order to ask whether these peptides were capable of binding to Drosophila PCNA, they were conjugated to streptavidin-agarose and incubated with the embryonic extracts. Following extensive washing, bound proteins were analysed by immunoblotting using an anti-Drosophila PCNA polyclonal antibody. The results in Figure Figure 4. PCNA-binding domain peptides specifically interact with PCNA from Drosophila embryonic extracts. Peptides conjugated to streptavidin-agarose beads were incubated with Drosophila embryonic extracts and the bound PCNA was analysed by SDS-PAGE followed by western blot analysis with a polyclonal rabbit antibody to Drosophila PCNA. The peptides are described in Materials and Methods and the numbered lanes show results as follows; lane 1, p21; lane 2, Dacapo; lane 3, Z50796; lane 4, Pogo; lane 5, Tigger; lane 6, Consensus; lane 7, Unrelated. (A) Western blot results of the PCNA bound to the beads following washing in PBS containing 0.05% Tween 20 and (B) the PCNA remaining in the supernatant following depletion. In order to investigate the relative affinities of these peptides for PCNA in an in vitro assay and to compare them with previously identified PCNA-binding peptides, they were tested for interaction with human PCNA in an ELISA-type assay. In these experiments, peptides were also tested in which the conserved glutamine (Q) was substituted by alanine (A). This residue is not only highly conserved, but in the human p21-derived peptide this mutation abrogates PCNA binding and the inhibition of DNA replication (12). The results in Figure Figure 5. ELISA analysis of peptide binding to PCNA. Biotinylated peptides were immobilized on ELISA plates pre-coated with streptavidin and incubated with purified human PCNA. Binding of PCNA to the immobilized peptides was assessed using the anti-PCNA polyclonal rabbit antiserum 3009, followed by HRP-conjugated anti-rabbit antibody and colorimetric detection at 450 nm. Dark blue bars represent binding to peptides derived from the wild-type protein sequence and light blue bars show results using the equivalent peptide with the conserved Q residue substituted by A. In order to examine whether these peptides are binding to the same region of PCNA as human p21, competition ELISA assays were performed. In these experiments, the ability of each peptide to bind to PCNA was tested in the presence of varying concentrations of the competing, non-biotinylated peptide KRRQTSMTDFYHSKRRLIFS, which is derived from the human p21 protein (Fig. Figure 6. Competition ELISA analysis of peptide-PCNA interactions. Various peptides (A, p21; B, Dacapo; C, Z50796; D, Consensus; E, Pogo; F, Tigger) were immobilized in individual wells and their capacity to bind PCNA in the presence of the non-biotinylated p21-derived peptide KRRQTSMTDFYHSKRRLIFS was tested by adding this peptide (closed squares) to the PCNA-containing solution immediately before addition to the ELISA plate wells. An unrelated peptide was used as a control for competition (open squares). The OD450 (arbitrary units, x-axis), indicating the amount of PCNA bound, is plotted against log10[competing peptide concentration in ng/ml] (y-axis). The p21-derived peptide is able to compete effectively for PCNA binding in each case, compared with the control peptide, strongly suggesting that the p21-derived peptide is binding the same site on PCNA as the immobilized peptide. Figure 7. A conserved PCNA-binding motif. PCNA-binding domains are shown in protein homologues of p21, Fen1, XPG, MCMT and Pogo transposase. The accession number is indicated in the second column where appropriate and the number in brackets in the third column indicates the position of the first amino acid residue shown (where known). Identical residues are highlighted in blue. Conserved protein motifs provide useful predictive tools for examining the large numbers of sequences available in the public domain databases. Proteins containing a consensus site can then be tested biochemically for the predicted interaction. In the PCNA-binding motif described here, only a subset of the residues are highly conserved. The BLAST and FASTA algorithms, which are commonly used for database searching, give an identical weighting to each residue of the search sequence, so matched sequences will not necessarily correspond to the consensus. Therefore, to search for best matches to the overall PCNA-binding consensus, we have used a profile search method, in which sequence alignments are used to construct an amino acid profile, which is then used to search protein sequence databases. The SWISSPROT database was searched using PROFILESEARCH with profiles derived from various combinations of the amino acid sequences shown in Figure Figure 8. Results of profile searching for PCNA-binding consensus motifs. Various combinations of the 20 amino acid sequences shown in Figure 7 were used to draw up profiles and these were used to search the SWISSPROT database (Materials and Methods for details). A selection of the best eukaryotic protein matches, excluding input sequences, are shown. All data shown here is described in the database entry for which the accession number is shown. Residues shown in blue are equivalent to those which have direct PCNA interactions in the p21-derived PCNA-binding region and which are highly conserved between the sequences shown in Figure 7. The function and subcellular localization of the majority of these proteins indicate that they are unlikely to interact with PCNA, with the notable exception of S.cerevisiae ScUNG (see text for discussion). One notable exception is the identification of a PCNA-binding motif in the uracil-DNA glycosylase (UNG) protein of S.cerevisiae (ScUNG), which gave a very high score in several profile searches. This protein functions in base excision repair (BER) to remove mutagenic uracil residues resulting from the deamination of cytosine in DNA (32). Recently, two forms of UNG protein have been identified in human and mouse, which result from alternative splicing events: Ung1 is targeted to mitochondria and Ung2, in which the N-terminal 44 amino acids are encoded by a previously unrecognized exon, is specifically targeted to the nucleus (Fig. Figure 9. Predicted PCNA binding motifs. (Top) Alignment of the N-terminal regions of uracil-DNA glycosylase (UNG) proteins from human, mouse and S.cerevisiae (33,62), showing that a PCNA-binding motif is conserved in the specifically nuclear forms of UNG in mouse and human and in the yeast protein. Ung1 and Ung2 proteins are aligned up to the common splice site (#) and the S.cerevisiae sequence is aligned beneath. Identical residues are shown in colour and the stars indicate the conserved residues of the predicted PCNA-binding motif. (Bottom) Alignment of the N-terminal regions of DNA ligase I homologues. The proteins shown are (accession no. in brackets) from human (M36067), mouse (P37913), Xenopus (P51892), S.pombe (P12000) and S.cerevisiae (P04819). As above, identical residues are shown in colour and the stars indicate the conserved residues of the predicted PCNA-binding motif. A number of the proteins described here are known by immunofluorescence studies to be targeted (like PCNA itself) to sites of DNA replication and/or repair (35-37; E.Warbrick, P.Coates and P.A.Hall, unpublished observations). We therefore searched for putative PCNA-binding domains in proteins known to be targeted to replication foci. In this way, a consensus PCNA-binding motif was found at the N-terminus of DNA ligase I, which plays an essential role in both DNA replication and nucleotide excision repair and is known to be targeted to replication foci (39,40). The predicted PCNA-binding motif is close to the N-terminus of the protein and is conserved in a wide range of eukaryotes (Fig. The p21/p27 family of CDK inhibitors are multifunctional proteins which contain several functional domains. The N-terminal region of these proteins contains domains that interact with CDK/cyclin complexes, which are central to the regulation of the cell cycle (45,46). Members of the p21 family are also capable of binding to PCNA through a region in the C-terminus and this domain is capable of inhibiting DNA replication in a PCNA-dependent manner (12,47,48). Although identified as cyclin-dependent kinase inhibitors, members of the p21/p27 family can also promote the assembly of CDKs and cyclins and p21 (at low molar ratios) can stimulate the kinase activity of these complexes (49). Though p21, p27 and p57 are all capable of directing accumulation of Cdk4 and cyclin D in the nucleus, p21 is particularly efficient and the C-terminal region of the molecule is required for this process. This region contains a bipartite nuclear localization signal which is coincident with the PCNA-binding domain, suggesting that this region may be important somehow for targeting p21 to specific sites within the nucleus. This issue is complicated, however, by the existence of an adjacent, though distinct, cyclin/CDK inhibitory domain in the C-terminus of p21 (50). An attractive model can be proposed in which, rather than representing the sole point of contact with PCNA, the PCNA-binding motif contained within p21, Fen1, XPG, MCMT, etc. is involved primarily in targeting the proteins to the DNA replication machinery within the nucleus. The Dacapo protein in D.melanogaster has been described as belonging to the p21/p27 family of cyclin-dependent kinase inhibitors (17,18). Here we describe identification of the protein by its ability to bind to PCNA, which places it in the same functional class as p21. Dacapo encodes an inhibitor of cyclin E/Cdk2 and is required to regulate cell cycle arrest of epidermal cells at the correct stage of embryogenesis. Unlike p21 in mammalian systems, Dacapo is required for normal embryonic development; loss of function mutants are lethal, with severe developmental defects due to overproliferation (17,18,51). Human p21 and Dacapo do not show a high overall homology (25% identity), rather, the homology is restricted to the regions required for CDK, cyclin and PCNA interactions. Within the PCNA-binding motif, it is predominantly those residues required for the interaction which are conserved (Fig. Pogo family transposons were first identified in Drosophila, where they are no longer active (19). They are classified as DNA or class II transposons which move by excision and re-integration into the genome, without an RNA intermediate. Such elements are flanked by terminal inverted repeats (TIRs) and encode a transposase protein which binds specifically to the TIRs and catalyses the cutting and pasting events which result in transposition. Mobility is dependent only upon the presence of the TIRs and a catalytically active transposase protein, so non-autonomous elements are as likely to be transposed as autonomous elements. Recently, a study of the molecular archaeology of human interspersed repeats with TIRs has revealed the presence of several groups with the characteristics of type II DNA-mediated transposons (24). These transposons are no longer mobile and have been described as `molecular fossils'. The human TIR-flanked repeats were analysed using consensus sequences from multiple alignments and one group showed significant homology to Pogo, both in the sequence of the TIRs and the coding sequence (24). This group contained two classes of element: Tigger1, so called as they represent a human Pogo (52), was primarily represented by full-length elements, of which there are an estimated 3000 copies in the human genome; Tigger2 is less well characterized and its coding sequence cannot be unambiguously defined, as it is less abundant and predominantly represented by internal deletion products. Here we describe the identification of a PCNA-binding motif in the Pogo transposase and find that this motif is conserved in the second ORF of the Tigger1 transposon. Equivalent domains from both proteins are functional in binding to both Drosophila and human PCNA. This evidence points to a specific function for the domain, which raises many interesting issues concerning the role of PCNA binding in transposase function. Since neither Tigger nor Pogo transposons appear to be capable of transposition, functional studies cannot be undertaken. However, it is tempting to speculate that the transposase may utilize the relatively open conformation of DNA around the replication fork to enhance integration and/or excision. Type II DNA transposons move by a cut-and-paste mechanism, so they do not proliferate unless excision is followed by gap repair or the element moves from a replicated to a still unreplicated part of the genome (53,54). This raises the intriguing possibility that the interaction of the Pogo/Tigger transposases with PCNA may be involved in targeting integration to unreplicated portions of the genome. Pogo contains two ORFs which are spliced to give a single ORF encoding a predicted protein of 499 amino acids. The identification of a functional PCNA-binding site in the second ORF of Tigger1 suggests that, as is the case in Pogo, a single Tigger1 transcript was spliced to encode a single polypeptide. Putative transposases in other organisms also show homology to Pogo and Tigger. The most highly related are the Pot2 and Fot1 fungal transposases, which do not appear to contain a consensus PCNA-binding domain, suggesting that an interaction with PCNA is not a feature of all members of this family of transposons (54,55). The Tigger and Pogo transposases also share homology with cellular proteins, for example Pdc1 in S.cerevisiae and its Kluyveromyces lactis homologue, Rag3, which are transcription factors, and the murine proteins Jerky and CENP-B, which is a eukaryotic centromere-associated protein (55-58). However, in these cellular proteins, conserved residues known to be involved in the transposase active site are mutated, suggesting that transposase gene-encoded proteins have been adapted to a cellular function. It is possible that the PCNA binding site in Pogo and Tigger transposases was derived from a cellular PCNA-binding protein through an excision event. The PCNA-binding motifs shown in Figure In the p21 peptide, the residues 139GRKRR143 are involved in poorly ordered interactions with the acidic C-terminus of PCNA (31). Human and mouse DNA ligase I homologues do not have any amino acids N-terminal of the conserved Q, suggesting that in these cases, there may not be an interaction with the C-terminus of PCNA. The C-terminal amino acids of the p21 peptide 152HSKRRLIFS160 form an antiparallel [beta]-sheet with the connector loop and these amino acids have been shown to be important in the inhibition of DNA replication by this peptide (12,31). In the Pogo-derived peptide, which has a high affinity for PCNA, these residues are absent. This indicates that the [beta]-sheet interaction with the interdomain linker region on PCNA is not essential. However, this linker domain is also involved in forming the hydrophobic pocket with which the X(h)XX(a)(a) motif interacts, so is still essential for the interaction, as confirmed by the results in Figure We present evidence that Dacapo and the transposase proteins encoded by Pogo and Tigger bind to the same site on PCNA as do p21 and Fen1, through a conserved QXX(h)XX(a)(a) motif. Further, we show that an entirely synthetic peptide can be designed based on consensus protein sequences which has a high affinity of binding to PCNA. However, a close examination of the PCNA-binding sequences suggests that the molecular interactions of the proteins described here are varied outside the core PCNA-binding domain. An analysis of the exact mechanisms of how these proteins interact with PCNA and how the interactions are coordinated and regulated will be important in understanding the role PCNA plays as a target for cell cycle regulation. We thank Steve Elledge for the two-hybrid constructs and library, Daryl Henderson for Drosophila PCNA cDNA and for anti-Drosophila PCNA antibodies, Nickolai Zhelev for purified human PCNA, Kathryn Ball for peptides, Nicole Gnadt for communicating results prior to publication and Álvaro Tavares for Drosophila embryonic extracts and helpful advice. This work was supported by grants from the Cancer Research Campaign and the Association for International Cancer Research.
Pogo and Dacapo proteins contain conserved consensus PCNA-binding domains
Use of the PCNA consensus as a tool for predicting PCNA-binding proteins
DISCUSSION
Dacapo is a member of the p21WAF1/Cip1 family of CDK inhibitors
The Pogo transposase interacts with PCNA
Molecular analysis of the PCNA interaction
Summary
ACKNOWLEDGEMENTS
REFERENCES
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