A Quantitative Study of the in Vitro Binding of the C-Terminal Domain of p21 to PCNA:  Affinity, Stoichiometry, and Thermodynamics

Zheleva, Daniella; Zhelev, Nikolai; Fischer, Peter M.; Duff, Susan V.; Warbrick, Emma; Blake, David G.; Lane, David P.

doi:10.1021/bi992498r

Cited by 83 publications

(108 citation statements)

References 48 publications

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“…It is likely that these short motifs are critical for the stability of the total binding interaction and must interact with complementary residues on ␤. Although the role and importance of additional contacts between ␤-binding proteins and ␤ is yet to be determined, it appears that they may be less important to core binding than for the PCNA-binding proteins (21,31,32). Indeed, whereas a tripeptide can inhibit the in vitro binding of ␣ and ␦ to ␤, and an octapeptide can inhibit phage T4 polymerase holoenzyme formation (33), at least several flanking amino acids are required in addition to the QxxLxxFF consensus motif for inhibition of binding to eukaryotic PCNA (32).…”

Section: Resultsmentioning

confidence: 99%

A universal protein–protein interaction motif in the eubacterial DNA replication and repair systems

Dalrymple

Kongsuwan

Wijffels

et al. 2001

Proc. Natl. Acad. Sci. U.S.A.

312

368

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The interaction between DNA polymerases and sliding clamp proteins confers processivity in DNA synthesis. This interaction is critical for most DNA replication machines from viruses and prokaryotes to higher eukaryotes. T he replication of DNA in eubacteria involves many proteins organized into a complex multifunctional machine termed the replisome. A central enzyme is the multisubunit DNA polymerase III holoenzyme. In Escherichia coli, and probably in most other eubacteria, the DnaE ortholog (␣ subunit) is in the core of the replicative polymerase, whereas in many Grampositive organisms a related enzyme, PolC, is proposed to have this function (1). The processivity of the polymerase is conferred by the direct interaction of the ␤ subunit (clamp protein) of DNA polymerase III (2, 3), with the DnaE (and presumably PolC) subunits. ␤ is loaded onto DNA by a clamp loader comprised of single ␦ and ␦Ј subunits and four ͞␥ subunits (1). The ␤ dimer thence encircles the DNA without actually binding to it. In addition to DnaE, three other E. coli DNA polymerases appear to interact with ␤. PolB (DNA polymerase II) is involved in DNA repair (4) and the addition of ␤ and the clamp loader increases its processivity in vitro (5, 6). Similarly, ␤ and the clamp loader together increase both the processivity (7) and efficiency (8) of DNA synthesis by DNA polymerase IV (DinB). ␤ also appears to play a similar role in the activity of DNA polymerase V (8) (the UmuDЈ 2 UmuC complex) and the UmuD subunit has been shown to bind to ␤ (9).Experimental evidence shows that at least some ␤-binding proteins can interact productively with ␤ from heterologous species. For example, PolC subunits from Staphylococcus aureus, Streptococcus pyogenes, and Bacillus subtilis can use E. coli ␤ as their processivity subunit (1, 10, 11). In contrast, E. coli DnaE cannot use ␤ from the other species (11), the E. coli clamp loader complex cannot load S. aureus ␤ (11), and the S. pyogenes clamp loader complex cannot load E. coli ␤ (1).In the absence of any experimentally identified ␤-binding sites in proteins, a bioinformatics approach was undertaken to identify putative ␤-binding motifs. The role of the putative motif was then examined by yeast two-hybrid and peptide-binding experiments with native and modified sequences. Materials and MethodsSources of Amino Acid Sequences. Amino acid sequences and alignments were derived from: PSI-BLAST of the protein database at the National Center for Biotechnology Information (NCBI), and BLAST of preliminary sequence data from NCBI at http:͞͞ www.ncbi.nlm.nih.gov͞Microb_blast͞unfinishedgenome.html, Institute for Genomic Research at http:͞͞www.tigr.org, Department of Energy Joint Genome Institute at http:͞͞spider.jgipsf.org͞JGI_microbial͞html͞, Sanger Center at http:͞͞ www.sanger.ac.uk͞DataSearch͞omniblast.shtml, and ERGO at http:͞͞wit.integratedgenomics.com͞IGwit͞. Alignments of available sequences of all members of eubacterial protein families known to bind to ␤ were compiled with manual editing in regions of variab...

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Section: Resultsmentioning

confidence: 99%

A universal protein–protein interaction motif in the eubacterial DNA replication and repair systems

Dalrymple

Kongsuwan

Wijffels

et al. 2001

Proc. Natl. Acad. Sci. U.S.A.

312

368

View full text Add to dashboard Cite

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“…The non-cooperative 1:1 stoichiometry is common to all PIP-box containing proteins whose binding to PCNA has been studied by calorimetry 19,29 . The mode of p15 binding to PCNA, however, differs importantly in that p15 extends its interaction site N-terminally from the PIP-box-binding groove on the front-face to the inner surface of the PCNA ring.…”

Section: Discussionmentioning

confidence: 99%

“…To study the role of different p15 regions in the interaction with PCNA, we designed three p15 fragments: the N-terminal deletion mutant p15 (hereafter named p15 DN ) lacking many of the residues experiencing reduced NMR signal intensity and enhanced 15 N R 2 rates on PCNA binding; the p15 fragment comprising the central residues that experience the largest drop in NMR signal intensity on PCNA binding; and p15 [59][60][61][62][63][64][65][66][67][68][69][70] that corresponds to the smallest PIP-box containing fragment of p21 with reported binding to PCNA 19 .…”

Section: P15-pcna Interaction Involves An Extended Pip-box Regionmentioning

confidence: 99%

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

et al. 2015

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The intrinsically disordered protein p15 PAF regulates DNA replication and repair by binding to the proliferating cell nuclear antigen (PCNA) sliding clamp. We present the structure of the human p15 PAF -PCNA complex. Crystallography and NMR show the central PCNA-interacting protein motif (PIP-box) of p15 PAF tightly bound to the front-face of PCNA. In contrast to other PCNA-interacting proteins, p15 PAF also contacts the inside of, and passes through, the PCNA ring. The disordered p15 PAF termini emerge at opposite faces of the ring, but remain protected from 20S proteasomal degradation. Both free and PCNA-bound p15 PAF binds DNA mainly through its histone-like N-terminal tail, while PCNA does not, and a model of the ternary complex with DNA inside the PCNA ring is consistent with electron micrographs. We propose that p15 PAF acts as a flexible drag that regulates PCNA sliding along the DNA and facilitates the switch from replicative to translesion synthesis polymerase binding.

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“…In addition, we could show that the proliferating cell nuclear antigen (PCNA), an auxiliary factor for DNA replication and repair, was co-immunoprecipitated with p21 in CD30-stimulated Karpas 299 cells. Association of p21 and PCNA (Zheleva et al, 2000) leads to inhibition of PCNA and can result in G1 and G2 cell cycle arrest (Cayrol et al, 1998). While the role of p21 in cell cycle arrest is generally accepted, controversial data were obtained considering its role in apoptotic cell death.…”

Section: Discussionmentioning

confidence: 99%

CD30-mediated cell cycle arrest associated with induced expression of p21CIP1/WAF1 in the anaplastic large cell lymphoma cell line Karpas 299

et al. 2001

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One of the major characteristics of anaplastic large cell lymphomas (ALCL) is the expression of the Ki-1/CD30 antigen. While the receptor mediates NF-kB-activation in Hodgkin's lymphomas, some data suggest the CD30-mediated apoptosis of other CD30-expressing cells. We were able to demonstrate that activation of CD30 leads to dierent eects regarding cell proliferation of the ALCL-derived cell lines Karpas 299 and JB6. Western and Northern blotting analysis revealed that CD30-induced growth inhibition of Karpas 299 cells correlated with a strong upregulation of the cell cycle inhibitor p21. We found a non activating point mutation at codon 273 in exon 8 of the p53 gene in Karpas 299 cells which indicates an p53-independent mechanism for induced p21 expression. Abundant p21 protein expression resulted in hypophosphorylation of the retinoblastoma protein (Rb) and inhibition of the proliferating cell nuclear antigen (PCNA). CD30-stimulated cells showed no indications of apoptotic cell death, like genomic DNA fragmentation or cleavage of the caspase-3 target protein poly (ADP-ribose) polymerase (PARP). Our results indicate that CD30 is able to mediate an p21-associated cell cycle arrest in ALCL with possible implications for prognosis and clinical treatment. Oncogene (2001) 20, 590 ± 598.

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A Quantitative Study of the in Vitro Binding of the C-Terminal Domain of p21 to PCNA: Affinity, Stoichiometry, and Thermodynamics

Cited by 83 publications

References 48 publications

A universal protein–protein interaction motif in the eubacterial DNA replication and repair systems

A universal protein–protein interaction motif in the eubacterial DNA replication and repair systems

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

CD30-mediated cell cycle arrest associated with induced expression of p21CIP1/WAF1 in the anaplastic large cell lymphoma cell line Karpas 299

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