A 39 Amino Acid Fragment of the Cell Cycle Regulator p21 Is Sufficient to Bind PCNA and Partially Inhibit DNA Replication in vivo

Abstract

The cell cycle regulator p21 interacts with and inhibits the DNA replication and repair factor proliferating cell nuclear antigen (PCNA). We have defined a 39 amino acid fragment of p21 which is sufficient to bind PCNA with high affinity (Kd 10-20 nM). This peptide can inhibit DNA replication in vitro and microinjection of a GST fusion protein containing this domain inhibited S phase in vivo. Despite its high affinity for PCNA, the free 39 amino acid peptide does not have a well-defined structure, as judged from circular dichroism and nuclear magnetic resonance measurements, suggesting an induced fit mechanism for the PCNA-p21 interaction. The association of the small peptide with PCNA was thermolabile, suggesting that portions of p21 adjoining the minimal region of contact stabilize the interaction. In addition, a domain containing 67 amino acids from the N-terminus of PCNA was defined as both necessary and sufficient for binding to p21.

Structure of p15PAF–PCNA complex and implications for clamp sliding during DNA replication and repair

“…Only the small and disordered p21 protein might have an affinity high enough to displace any other ligand from PCNA, explaining its dominant inhibition of DNA replication and cell cycle progression 31 . For instance, a C-terminal p21 fragment 32 binds PCNA with two orders of magnitude higher affinity than p15. Conversely, the affinity of PCNA for p15 is higher than for a PIPbox fragment of the p66 subunit of the replicative polymerase d (Pold; K d ¼ 15.4 mM, measured by ITC at 30°C) 29 , and similar to the translesion synthesis polymerase Z (TLS PolZ; K d ¼ 0.4 mM, measured by surface plasmon resonance at 25°C) 33 .…”

Proliferating Cell Nuclear Antigen as the Cell Cycle Sensor for an HLA-Derived Peptide Blocking T Cell Proliferation

“…A yeast two-hybrid screen identified PCNA as the cellular ligand for the DQ65-79 peptide, and this interaction was further confirmed by in vitro biochemistry and electron microscopy. Synthetic peptides derived from p21 interact with PCNA to block replication in vitro but have no effect on DNA synthesis in intact cells (12), most likely because they are not able to penetrate the cell membrane. In contrast, the DQ peptide interacts with PCNA in T cell nuclei when added to cells in culture and is highly anti-proliferative (6).…”

“…12 and this study; circular dichroism data not shown), while the DQ peptide has a strongly ␣ helical profile (data not shown). In addition, although both peptides can interact with PCNA, their mechanisms of inhibitory action are different as the p21 peptide binds to PCNA to block pol ␦-dependent replication (7,12) while the DQ65-79 peptide does not.…”

“…PCNA interacts with DNA pol ␦ and replication factor C to form a processive DNA polymerase complex (9 -11). Synthetic peptides corresponding to p21, a cell cycle inhibitor, bind PCNA and block the function of PCNA in the DNA polymerase complex in vitro (12). To determine whether DQ65-79 affects DNA replication by direct binding to PCNA, peptides were 1 -added to an in vitro system that measures pol ␦-dependent DNA synthesis (7).…”

Serine 123 Phosphorylation Modulates p21 Protein Stability and Activity by Suppressing Ubiquitin-independent Proteasomal Degradation

“…Second, through its carboxyl terminus, p21 is able to associate with proliferating cell nuclear antigen (PCNA) and inhibits the interaction of PCNA with replication factor (8), DNA polymerase ␦ (9, 10), and FEN1 (11,12), leading to inhibition of DNA synthesis (13,14).…”