Emma Warbrick

PCNA BINDING THROUGH A CONSERVED MOTIF

Proliferating cell nuclear antigen (PCNA) has recently been identified as a target for the binding of several proteins. The cell cycle regulatory protein, p21, and the replication endonuclease, Fen1, have already been described as competing for PCNA binding. Two recent reports have identified DNA (cytosine-5)methyltransferase (MCMT) and the DNA repair endonuclease XPG as binding to PCNA. The remarkable thing about these interactions is that they all seem to occur through a conserved motif that is likely to contact the same site on PCNA. This has fascinating implications for a regulatory network linking these diverse protein functions.

Post‐replicative base excision repair in replication foci

Base excision repair (BER) is initiated by a DNA glycosylase and is completed by alternative routes, one of which requires proliferating cell nuclear antigen (PCNA) and other proteins also involved in DNA replication. We report that the major nuclear uraci‐DNA glycosylase (UNG2) increases in S phase, during which it co‐localizes with incorporated BrdUrd in replication foci. Uracil is rapidly removed from replicatively incorporated dUMP residues in isolated nuclei. Neutralizing antibodies to UNG2 inhibit this removal, indicating that UNG2 is the major uraci‐DNA glycosylase responsible. PCNA and replication protein A (RPA) co‐localize with UNG2 in replication foci, and a direct molecular interaction of UNG2 with PCNA (one binding site) and RPA (two binding sites) was demonstrated using two‐hybrid assays, a peptide SPOT assay and enzyme‐linked immunosorbent assays. These results demonstrate rapid post‐replicative removal of incorporated uracil by UNG2 and indicate the formation of a BER complex that contains UNG2, RPA and PCNA close to the replication fork.

A small peptide inhibitor of DNA replication defines the site of interaction between the cyclin-dependent kinase inhibitor p21WAF1 and proliferating cell nuclear antigen

BACKGROUND p21WAF1 is a potent inhibitor of the cell-cycle regulatory cyclin-dependent kinases (Cdks). It acts on Cdks in the G1 and S phases of the cell cycle, and also binds to proliferating cell nuclear antigen (PCNA), blocking DNA replication in vitro. Transcription of p21WAF1 can be induced by the human tumour suppressor protein p53, suggesting that the action of p21WAF1 may be important in cancer prevention. We have investigated the interaction between p21WAF1 and PCNA using a genetic two-hybrid screen and with arrays of synthetic peptides derived from the p21WAF1 protein sequence. RESULTS We have established that the carboxy-terminal region of p21WAF1 interacts with PCNA in a yeast two-hybrid screen. Interaction with p21WAF1 involves the central loop of PCNA, which connects the two domains of the PCNA monomer. The interaction was finely mapped using peptides derived from the entire sequence of the p21WAF1 protein, and the critical residues were found to be QTSMTDFY (amino acids 144-151 of p21WAF1). Remarkably, a 20-residue peptide containing this sequence inhibited replication of simian virus 40 (SV40) DNA in vitro and could capture PCNA from whole cell extracts, demonstrating that small molecules can retain the biological activity characteristic of the whole protein. Sequential alanine-scan mutations of the peptide demonstrated that its ability to block replication correlates with its affinity for binding PCNA. CONCLUSIONS We have shown that PCNA and the cell-cycle regulator p21WAF1 interact in vivo, and that this interaction requires the central loop of PCNA and an eight amino-acid motif from the carboxyl terminus of p21WAF1.(ABSTRACT TRUNCATED AT 250 WORDS)

Homologous regions of Fen1 and p21Cip1 compete for binding to the same site on PCNA : a potential mechanism to co-ordinate DNA replication and repair

Following genomic damage, the cessation of DNA replication is co-ordinated with onset of DNA repair; this co-ordination is essential to avoid mutation and genomic instability. To investigate these phenomena, we have analysed proteins that interact with PCNA, which is required for both DNA replication and repair. One such protein is p21Cip1, which inhibits DNA replication through its interaction with PCNA, while allowing repair to continue. We have identified an interaction between PCNA and the structure specific nuclease, Fen1, which is involved in DNA replication. Deletion analysis suggests that p21Cip1 and Fen1 bind to the same region of PCNA. Within Fen1 and its homologues a small region (10 amino acids) is sufficient for PCNA binding, which contains an 8 amino acid conserved PCNA-binding motif. This motif shares critical residues with the PCNA-binding region of p21Cip1. A PCNA binding peptide from p21Cip1 competes with Fen1 peptides for binding to PCNA, disrupts the Fen1-PCNA complex in replicating cell extracts, and concomitantly inhibits DNA synthesis. Competition between homologous regions of Fen1 and p21Cip1 for binding to the same site on PCNA may provide a mechanism to co-ordinate the functions of PCNA in DNA replication and repair.

A lack of premature termination codon read-through efficacy of PTC124 (Ataluren) in a diverse array of reporter assays.

The drug molecule PTC124 (Ataluren) has been described as a read-through agent, capable of suppressing premature termination codons (PTCs) and restoring functional protein production from genes disrupted by nonsense mutations. Following the discovery of PTC124 there was some controversy regarding its mechanism of action with two reports attributing its activity to an off-target effect on the Firefly luciferase (FLuc) reporter used in the development of the molecule. Despite questions remaining as to its mechanism of action, development of PTC124 continued into the clinic and it is being actively pursued as a potential nonsense mutation therapy. To thoroughly test the ability of PTC124 to read through nonsense mutations, we conducted a detailed assessment comparing the efficacy of PTC124 with the classical aminoglycoside antibiotic read-through agent geneticin (G418) across a diverse range of in vitro reporter assays. We can confirm the off-target FLuc activity of PTC124 but found that, while G418 exhibits varying activity in every read-through assay, there is no evidence of activity for PTC124.

Essential interaction between the fission yeast DNA polymerase δ subunit Cdc27 and Pcn1 (PCNA) mediated through a C-terminal p21Cip1-like PCNA binding motif

Direct interaction between DNA polymerase δ and its processivity factor proliferating cell nuclear antigen (PCNA) is essential for effective replication of the eukaryotic genome, yet the precise manner by which this occurs is unclear. We show that the 54 kDa subunit of DNA polymerase δ from Schizosaccharomyces pombe interacts directly with Pcn1 (PCNA) both in vivo and in vitro. Binding is effected via a short sequence at the C‐terminus of Cdc27 with significant similarity to the canonical PCNA binding motif first identified in the mammalian p21Cip1 protein. This motif is both necessary and sufficient for binding of Pcn1 by Cdc27 in vitro and is essential for Cdc27 function in vivo. We also show that the Pcn1 binding motif in Cdc27 is distinct from its binding site for Cdc1, the 55 kDa B‐subunit of polymerase δ, and present evidence that Cdc27 can bind to Pcn1 and Cdc1 simultaneously. Finally, we show that Cdc27 performs at least two distinct essential functions, one of which is independent of Pcn1 binding.

Fen1 expression: a novel marker for cell proliferation

The identification of antigens whose expression is associated with the cell cycle is a particularly attractive method with which to define proliferative populations in histological and cytological preparations. A polyclonal antibody 3220 has been raised which recognizes the structure‐specific endonuclease Fen1 and can be used for a wide range of applications including western blotting, immunoprecipitation and immunohistochemical analysis. This antibody has been used to examine Fen1 levels by immunoblotting and its subcellular localization in cultured cells and tissue samples by immunostaining. Although the role Fen1 plays in DNA replication has been well characterized, its function in DNA repair is not so clear. The possible roles of Fen1 in repair have been investigated by examining any changes in level or localization of Fen1 in response to DNA damaging agents. We find that Fen1 is a nuclear antigen, that it is expressed by cycling cells, and that it co‐localizes with PCNA and polymerase α during S phase. Fen1 expression is topologically regulated in vivo and is associated with proliferative populations. No change has been found in either patterns or levels of Fen1 expression induced by DNA damaging agents, either in vivo or in vitro.

A functional analysis of PCNA-binding peptides derived from protein sequence, interaction screening and rational design

Proliferating cell nuclear antigen (PCNA) has no intrinsic enzymatic function, but functions as a sliding platform to mediate protein interactions with the DNA strand. Many proteins interact with PCNA through a small conserved motif with consensus QxxLxxFF. This work uses Schizosaccharomyces pombe and human cells to analyse the function of PCNA-binding peptides. Interacting peptides were identified using two-hybrid screening; one (pep102) binds directly to a physiologically relevant site on PCNA. The EGFP-pep102 overexpression phenotype is consistent with competitive blocking of PCNA–protein interactions. Various PCNA-binding peptides were all shown to inhibit PCNA function by competitive binding in both human and S. pombe cells as EGFP fusion proteins. The action of a p21(WAF1/Cip1)-derived peptide was complicated by the presence of additional functional domains and possible post-translational modification. The activity of pep102 was hampered by low expression in both model systems. The peptide derived from rational design (con1) was stable, highly active in inhibiting PCNA function both S. pombe and human cells and showed a high affinity for PCNA both in vitro and in vivo. These results validate the use of functional screening in yeast to identify peptide aptamers that are functional in mammalian cells; such aptamers provide excellent leads for small molecule antiproliferative therapies.

Two's company, three's a crowd: the yeast two hybrid system for mapping molecular interactions

I would like to thank all of my colleagues for their helpful suggestions, and to acknowledge the support of the Cancer Research Campaign.

An in vivo analysis of the localisation and interactions of human p66 DNA polymerase δ subunit

BackgroundDNA polymerase δ is essential for eukaryotic DNA replication and also plays a role in DNA repair. The processivity of this polymerase complex is dependent upon its interaction with the sliding clamp PCNA and the polymerase-PCNA interaction is largely mediated through the p66 polymerase subunit. We have analysed the interactions of the human p66 DNA polymerase δ subunit with PCNA and with components of the DNA polymerase δ complex in vivo.ResultsUsing the two-hybrid system, we have mapped the interaction domains for binding to the p50 polymerase δ subunit and with PCNA to the N-terminus and the C-terminus of p66, respectively. Co-immunoprecipitation experiments confirm that these interaction domains are functional in vivo. Expression of EGFP-p66 shows that it is a nuclear protein which co-localises with PCNA throughout the cell cycle. p66 is localised to sites of DNA replication during S phase and to repair foci following DNA damage. We have identified a functional nuclear localisation sequence and shown that localisation to replication foci is not dependent upon active nuclear import. Sub-domains of p66 act as dominant negative suppressors of colony formation, suggesting that p66 forms an essential structural link between the p50 subunit and PCNA. Analysis of the C-terminal PCNA binding motif shows that deletion of the QVSITGFF core motif results in a reduced affinity for PCNA, while deletion of a further 20 amino acids completely abolishes the interaction. A reduced affinity for PCNA correlates with reduced targeting to replication foci. We have confirmed the p66-PCNA interaction in vivo using fluorescence resonance energy transfer (FRET) techniques.ConclusionWe have defined the regions of p66 required for its interaction with PCNA and the p50 polymerase subunit. We demonstrate a functional link between PCNA interaction and localisation to replication foci and show that there is a direct interaction between p66 and PCNA in living cells during DNA replication. The dominant negative effect upon growth resulting from expression of p66 sub-domains confirms that the p66-PCNA interaction is essential in vivo.