Proliferating Cell Nuclear Antigen Binding Motif Research Articles

Checkpoint activation by Spd1: a competition-based system relying on tandem disordered PCNA binding motifs.

DNA regulation, replication and repair are processes fundamental to all known organisms and the sliding clamp proliferating cell nuclear antigen (PCNA) is central to all these processes. S-phase delaying protein 1 (Spd1) from S. pombe, an intrinsically disordered protein that causes checkpoint activation by inhibiting the enzyme ribonucleotide reductase, has one of the most divergent PCNA binding motifs known. Using NMR spectroscopy, in vivo assays, X-ray crystallography, calorimetry, and Monte Carlo simulations, an additional PCNA binding motif in Spd1, a PIP-box, is revealed. The two tandemly positioned, low affinity sites exchange rapidly on PCNA exploiting the same binding sites. Increasing or decreasing the binding affinity between Spd1 and PCNA through mutations of either motif compromised the ability of Spd1 to cause checkpoint activation in yeast. These results pinpoint a role for PCNA in Spd1-mediated checkpoint activation and suggest that its tandemly positioned short linear motifs create a neatly balanced competition-based system, involving PCNA, Spd1 and the small ribonucleotide reductase subunit, Suc22R2. Similar mechanisms may be relevant in other PCNA binding ligands where divergent binding motifs so far have gone under the PIP-box radar.

The Amino-Terminus of Mouse DNA Methyltransferase 1 Forms an Independent Domain and Binds to DNA with the Sequence Involving PCNA Binding Motif

DNA methylation patterns in genome are maintained during replication by a DNA methyltransferase Dnmt1. Mouse Dnmt1 is a 180 kDa protein comprising the N-terminal regulatory domain, which covers 2/3 of the molecule, and the rest C-terminal catalytic domain. In the present study, we demonstrated that the limited digestion of full-length Dnmt1 with different proteases produced a common N-terminal fragment, which migrated along with Dnmt1 (1-248) in SDS-polyacrylamide gel electrophoresis. Digestion of the N-terminal domains larger than Dnmt1 (1-248) with chymotrypsin again produced the fragment identical to the size of Dnmt1 (1-248). These results indicate that the N-terminal domain of 1-248 forms an independent domain. This N-terminal domain showed DNA binding activity, and the responsible sequence was narrowed to the 79 amino acid residues involving the proliferating cell nuclear antigen (PCNA) binding motif. The DNA binding activity did not distinguish between DNA methylated and non-methylated states, but preferred to bind to the minor groove of AT-rich sequence. The DNA binding activity of the N-terminal domain competed with the PCNA binding. We propose that DNA binding activity of the N-terminal domain contributes to the localization of Dnmt1 to AT-rich sequence such as Line 1, satellite, and the promoter of tissue-specific silent genes.

REV1 Protein Interacts with PCNA: Significance of the REV1 BRCT Domain In Vitro and In Vivo

REV1 protein, a eukaryotic member of the Y family of DNA polymerases, is involved in the tolerance of DNA damage by translesion DNA synthesis. It is unclear how REV1 is recruited to replication foci in cells. Here, we report that mouse REV1 can bind directly to PCNA and that monoubiquitylation of PCNA enhances this interaction. The interaction between REV1 protein and PCNA requires a functional BRCT domain located near the N terminus of the former protein. Deletion or mutational inactivation of the BRCT domain abolishes the targeting of REV1 to replication foci in unirradiated cells, but not in UV-irradiated cells. In vivo studies in both chicken DT40 cells and yeast directly support the requirement of the BRCT domain of REV1 for cell survival and DNA damage-induced mutagenesis.

PCNA Is a Cofactor for Cdt1 Degradation by CUL4/DDB1-mediated N-terminal Ubiquitination

Cdt1, a protein essential in G1 for licensing of origins for DNA replication, is inhibited in S-phase, both by binding to geminin and degradation by proteasomes. Cdt1 is also degraded after DNA damage to stop licensing of new origins until after DNA repair. Phosphorylation of Cdt1 by cyclin-dependent kinases promotes its binding to SCF-Skp2 E3 ubiquitin ligase, but the Cdk2/Skp2-mediated pathway is not essential for the degradation of Cdt1. Here we show that the N terminus of Cdt1 contains a second degradation signal that is active after DNA damage and in S-phase and is dependent on the interaction of Cdt1 with proliferating cell nuclear antigen (PCNA) through a PCNA binding motif. The degradation involves N-terminal ubiquitination and requires Cul4 and Ddb1 proteins, components of an E3 ubiquitin ligase implicated in protein degradation after DNA damage. Therefore PCNA, the matchmaker for many proteins involved in DNA and chromatin metabolism, also serves to promote the targeted degradation of associated proteins in S-phase or after DNA damage.

Cloning of two rat PDIP1 related genes and their interactions with proliferating cell nuclear antigen

Human polymerase delta-interacting protein 1 (PDIP1) is a tumor necrosis factor alpha and interleukin 6 inducible protein that interacts directly with proliferating cell nuclear antigen (PCNA) and the small subunit (p50) of DNA polymerase delta. PDIP1 binds PCNA and p50 simultaneously and stimulates polymerase delta activity in vitro in the presence, but not the absence, of PCNA. It has been suggested that PDIP1 provides a link between cytokine activation and DNA replication in eukaryotes. Here these authors report the cloning of two rat genes homologous to human PDIP1, termed rat PDIP1 and rat tumor necrosis factor-induced protein 1 (TNFAIP1). The rat PDIP1 is mapped to chromosome 1q36 cM region, spans approximately 18.7 kb, and is organized into six exons. The rat TNFAIP1 gene is mapped to chromosome 10q25 cM, spans approximately 12.9 kb, and is composed of seven exons. The deduced proteins of rat PDIP1 and rat TNFAIP1 share 63.1% sequence identity with each other and are highly conserved in the majority of the middle portion of the two proteins, which encode a BTB/POZ domain at the N-terminus and a PCNA binding motif (QTKV-EFP) at the C-terminus, respectively. The BTB / POZ domain and the PCNA binding motif are highly conserved during the evolution. Both rat PDIP1 and rat TNFAIP1 were demonstrated to interact with PCNA via BIAcore, GST pull-down, and co-immunoprecipitation assays. Like the human PDIP1, both rat PDIP1 and rat TNFAIP1 stimulate polymerase delta activity in vitro in a PCNA-dependent way.

PCNA clamp facilitates action of DNA cytosine methyltransferase 1 on hemimethylated DNA.

Proliferating cell nuclear antigen (PCNA) is a ring-shaped protein known as a processivity factor of DNA polymerase delta. In addition to this role, PCNA interacts with a number of other proteins to increase their local concentration at replicated DNA sites. DNA cytosine methyltransferase 1 (Dnmt1), which preserves epigenetic signals by completing the methylation of hemimethylated DNA after DNA replication, has been indicated as one of these PCNA binding proteins by a previous work. However, the molecular mechanisms and functional significance of their association have not yet been studied. Dnmt1 can be readily isolated from nuclear extracts by PCNA affinity chromatography. Studies of the interactions between the two proteins demonstrate that the N-terminal region of Dnmt1, which contains a typical PCNA binding motif, has core PCNA binding activity, and that the remaining portion of the protein exerts a negative influence on the interaction of Dnmt1 with PCNA. The affinity of Dnmt1 for DNA is much higher for DNA bound by PCNA than for free DNA. Furthermore, DNA methylation assays with hemimethylated DNA as a substrate revealed that PCNA clamp-bound DNA is methylated more efficiently by Dnmt1 than is free DNA. These results provide the first biochemical evidence that physical interactions between PCNA and Dnmt1 facilitate the methylation of newly neplicated DNA, on which PCNA remains associated as a functional clamp.

Physical interaction between proliferating cell nuclear antigen and replication factor C from Pyrococcus furiosus.

Proliferating cell nuclear antigen (PCNA), which is recognized as a DNA polymerase processivity factor, has direct interactions with various proteins involved in the important genetic information processes in Eukarya. We determined the crystal structure of PCNA from the hyperthermophilic archaeon, Pyrococcus furiosus (PfuPCNA) at 2.1 A resolution, and found that the toroidal ring-shaped structure, which consists of homotrimeric molecules, is highly conserved between the Eukarya and Archaea. This allowed us to examine its interaction with the loading factor at the atomic level. The replication factor C (RFC) is known as the loading factor of PCNA on to the DNA strand. P. furiosus RFC (PfuRFC) has a PCNA binding motif (PIP-box) at the C-terminus of the large subunit (RFCL). An 11 residue-peptide containing a PIP-box sequence of RFCL inhibited the PCNA-dependent primer extension ability of P. furiosus PolI in a concentration-dependent manner. To understand the molecular interaction mechanism of PCNA with PCNA binding proteins, we solved the crystal structure of PfuPCNA complexed with the PIP-box peptide. The interaction mode of the two molecules is remarkably similar to that of human PCNA and a peptide containing the PIP-box of p21(WAF1/CIP1). Moreover, the PIP-box binding may have some effect on the stability of the ring structure of PfuPCNA by some domain shift. Our structural analysis on PfuPCNA suggests that the interaction mode of the PIP-box with PCNA is generally conserved among the PCNA interacting proteins and that the functional meaning of the interaction via the PIP-box possibly depends on each protein. A movement of the C-terminal region of the PCNA monomer by PIP-box binding may cause the PCNA ring to be more rigid, suitable for its functions.

Physical and functional interactions of human DNA polymerase eta with PCNA.

Human DNA polymerase eta (hPoleta) functions in the error-free replication of UV-damaged DNA, and mutations in hPoleta cause cancer-prone syndrome, the variant form of xeroderma pigmentosum. However, in spite of its key role in promoting replication through a variety of distorting DNA lesions, the manner by which hPoleta is targeted to the replication machinery stalled at a lesion site remains unknown. Here, we provide evidence for the physical interaction of hPoleta with proliferating cell nuclear antigen (PCNA) and show that mutations in the PCNA binding motif of hPoleta inactivate this interaction. PCNA, together with replication factor C and replication protein A, stimulates the DNA synthetic activity of hPoleta, and steady-state kinetic studies indicate that this stimulation accrues from an increase in the efficiency of nucleotide insertion resulting from a reduction in the apparent K(m) for the incoming nucleotide.

P15(PAF), a novel PCNA associated factor with increased expression in tumor tissues.

Proliferating cell nuclear antigen (PCNA) is an essential protein in both DNA replication and DNA damage repair. A novel 15 kD protein, p15(PAF), was identified as a PCNA-associated factor in a yeast two-hybrid screen using PCNA as the bait. p15(PAF) is localized primarily in the nucleus. p15(PAF) shares the conserved PCNA binding motif with several other PCNA binding proteins including CDK inhibitor p21. Overexpression of p15(PAF) competes with p21-PCNA binding. Mutation of this motif in p15(PAF) abolished its PCNA-binding activity. Notably, p15(PAF) expression in several types of tumor tissues was significantly increased, especially in esophageal tumors. Like PCNA, p15(PAF) may possess prognostic significance in a broad array of human cancers.

Functional Interaction of Proliferating Cell Nuclear Antigen with MSH2-MSH6 and MSH2-MSH3 Complexes

Eukaryotic DNA mismatch repair requires the concerted action of several proteins, including proliferating cell nuclear antigen (PCNA) and heterodimers of MSH2 complexed with either MSH3 or MSH6. Here we report that MSH3 and MSH6, but not MSH2, contain N-terminal sequence motifs characteristic of proteins that bind to PCNA. MSH3 and MSH6 peptides containing these motifs bound PCNA, as did the intact Msh2-Msh6 complex. This binding was strongly reduced when alanine was substituted for conserved residues in the motif. Yeast strains containing alanine substitutions in the PCNA binding motif of Msh6 or Msh3 had elevated mutation rates, indicating that these interactions are important for genome stability. When human MSH3 or MSH6 peptides containing the PCNA binding motif were added to a human cell extract, mismatch repair activity was inhibited at a step preceding DNA resynthesis. Thus, MSH3 and MSH6 interactions with PCNA may facilitate early steps in DNA mismatch repair and may also be important for other roles of these eukaryotic MutS homologs.

Retroviral delivery of peptide modulators of cellular functions.

Stable transduction of genetic material, in combination with sensitive methodologies for in vivo study of cell physiology, provides an opportunity to efficiently evaluate the functions of regulatory proteins. To dissect the minimal therapeutic function of such proteins, we have stably expressed protein microdomains as fusions, composed of short peptides, and detected specific subfunctions distinct from holoprotein function, using flow cytometry and other techniques. We demonstrate that retroviral delivery of the 24-amino-acid proliferating cell nuclear antigen-binding motif (p21C), derived from the C-terminus of the cell cycle inhibitor protein, p21, is sufficient to induce cell cycle arrest. Cells expressing this peptide motif reversibly execute both G1- and G2-checkpoint controls that are normally activated subsequent to interference with DNA synthesis. The p21C effect is distinct from results obtained with an intact p21 protein that also binds cyclin-CDK complexes and arrested cells exclusively at the G1/S transition. Thus, microdomains can exert unique biological effects compared to the parental molecules from which they were derived. To further evaluate the peptide delivery strategy, we analyzed the role of various kinases in IgE-mediated stimulation of mast cell exocytosis. Primary bone marrow-derived mast cells were transduced with retroviral constructs encoding short-kinase inhibitor motifs and analyzed by flow cytometry for effects on exocytosis. We found that a specific protein kinase A (PKA) inhibitor peptide suppressed IgE-mediated stimulation of mast cell exocytosis. This anti-exocytotic effect was mimicked by a small molecule inhibitor of PKA (KT5720). Thus, the ability to express protein microdomains can be a powerful means to subtly perturb cellular physiology in manners that reveal new paths for therapeutic intervention. We believe that such approaches might allow for new forms of gene therapy to become available.

Essential interaction between the fission yeast DNA polymerase delta subunit Cdc27 and Pcn1 (PCNA) mediated through a C-terminal p21(Cip1)-like PCNA binding motif.

Direct interaction between DNA polymerase delta 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 delta 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 p21(Cip1) 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 delta, 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.

Direct Interaction of Proliferating Cell Nuclear Antigen with the p125 Catalytic Subunit of Mammalian DNA Polymerase δ

The formation of a complex between DNA polymerase delta (pol delta) and its sliding clamp, proliferating cell nuclear antigen (PCNA), is responsible for the maintenance of processive DNA synthesis at the leading strand of the replication fork. In this study, the ability of the p125 catalytic subunit of DNA polymerase delta to engage in protein-protein interactions with PCNA was established by biochemical and genetic methods. p125 and PCNA were shown to co-immunoprecipitate from either calf thymus or HeLa extracts, or when they were ectopically co-expressed in Cos 7 cells. Because pol delta is a multimeric protein, this interaction could be indirect. Thus, rigorous evidence was sought for a direct interaction of the p125 catalytic subunit and PCNA. To do this, the ability of recombinant p125 to interact with PCNA was established by biochemical means. p125 co-expressed with PCNA in Sf9 cells was shown to form a physical complex that can be detected on gel filtration and that can be cross-linked with the bifunctional cross-linking agent Sulfo-EGS (ethylene glycol bis (sulfosuccinimidylsuccinate)). An interaction between p125 and PCNA could also be demonstrated in the yeast two hybrid system. Overlay experiments using biotinylated PCNA showed that the free p125 subunit interacts with PCNA. The PCNA overlay blotting method was also used to demonstrate the binding of synthetic peptides corresponding to the N2 region of pol delta and provides evidence for a site on pol delta that is involved in the protein-protein interactions between PCNA and pol delta. This region contains a sequence that is a potential member of the PCNA binding motif found in other PCNA-binding proteins. These studies provide an unequivocal demonstration that the p125 subunit of pol delta interacts with PCNA.

The C-terminal Region of p21SDI1/WAF1/CIP1 Is Involved in Proliferating Cell Nuclear Antigen Binding but Does Not Appear to Be Required for Growth Inhibition

The cyclin-dependent kinase (Cdk) inhibitor p21SDI1/WAF1/CIP1 has been found to be involved in cell senescence, cell cycle arrest, and differentiation. p21SDI1 inhibits the activity of several Cdks, in contrast to other inhibitors such as p15INK4B and p16INK4A, which act on specific cyclin-Cdk complexes. Of interest were reports that p21SDI1 also bound proliferating cell nuclear antigen (PCNA), an auxiliary protein for DNA polymerase delta, and inhibited DNA replication but not DNA repair in vitro. To better understand the function of this interaction in vivo, we first determined the region of p21SDI1 that was needed for PCNA binding. Analysis of deletion mutants of p21SDI1, which covered the majority of the protein, revealed that deletion of either amino acids 142-147 or 149-154 resulted in loss of ability to bind a glutathione S-transferase-PCNA fusion protein. Site-directed mutagenesis in this region led to the identification of the PCNA binding motif RQXXMTXFYXXXR and demonstrated that mutation of either amino acid Met-147 or Phe-150 resulted in almost complete ablation of PCNA binding. Interestingly, when we determined DNA synthesis inhibitory activity of deletion mutants or point mutants that were unable to bind Cdk2 and/or PCNA, we found that loss of binding to PCNA did not affect inhibitory activity, whereas lack of Cdk2 binding greatly reduced the same. This result suggests that the primary mechanism for inhibition of DNA synthesis by p21SDI1 occurs via inhibition of Cdk activity.