Structure of proliferating cell nuclear antigen (PCNA) bound to an APIM peptide reveals the universality of PCNA interaction

Proliferating cell nuclear antigen (PCNA) provides a molecular platform for numerous protein–protein interactions in DNA metabolism. A large number of proteins associated with PCNA have a well characterized sequence termed the PCNA-interacting protein box motif (PIPM). Another PCNA-interacting sequence termed the AlkB homologue 2 PCNA-interacting motif (APIM), comprising the five consensus residues (K/R)-(F/Y/W)-(L/I/V/A)-(L/I/V/A)-(K/R), has also been identified in various proteins. In contrast to that with PIPM, the PCNA–APIM interaction is less well understood. Here, the crystal structure of PCNA bound to a peptide carrying an APIM consensus sequence, RFLVK, was determined and structure-based interaction analysis was performed. The APIM peptide binds to the PIPM-binding pocket on PCNA in a similar way to PIPM. The phenylalanine and leucine residues within the APIM consensus sequence and a hydrophobic residue that precedes the APIM consensus sequence are crucially involved in interactions with the hydrophobic pocket of PCNA. This interaction is essential for overall binding. These results provide a structural basis for regulation of the PCNA interaction and might aid in the development of specific inhibitors of this interaction.

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Protein-protein interactions of yeast DNA polymerase III with mammalian and yeast proliferating cell nuclear antigen (PCNA)/cyclin

Biochimica et Biophysica Acta (BBA) - Gene Structure and Expression ◽

10.1016/0167-4781(88)90097-8 ◽

1988 ◽

Vol 951 (2-3) ◽

pp. 274-279 ◽

Cited By ~ 11

Author(s):

Glenn A. Bauer ◽

Peter M.J. Burgers

Keyword(s):

Dna Polymerase ◽

Protein Interactions ◽

Proliferating Cell Nuclear Antigen ◽

Nuclear Antigen ◽

Dna Polymerase Iii ◽

Protein Protein Interactions ◽

Polymerase Iii ◽

Proliferating Cell

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Maneuvers on PCNA Rings during DNA Replication and Repair

Genes ◽

10.3390/genes9080416 ◽

2018 ◽

Vol 9 (8) ◽

pp. 416 ◽

Cited By ~ 23

Author(s):

Dea Slade

Keyword(s):

Dna Replication ◽

Protein Interactions ◽

Proliferating Cell Nuclear Antigen ◽

Binding Proteins ◽

Nuclear Antigen ◽

Vast Number ◽

Post Translational Modifications ◽

Dna Replication And Repair ◽

Replicative Polymerases ◽

Proliferating Cell

DNA replication and repair are essential cellular processes that ensure genome duplication and safeguard the genome from deleterious mutations. Both processes utilize an abundance of enzymatic functions that need to be tightly regulated to ensure dynamic exchange of DNA replication and repair factors. Proliferating cell nuclear antigen (PCNA) is the major coordinator of faithful and processive replication and DNA repair at replication forks. Post-translational modifications of PCNA, ubiquitination and acetylation in particular, regulate the dynamics of PCNA-protein interactions. Proliferating cell nuclear antigen (PCNA) monoubiquitination elicits ‘polymerase switching’, whereby stalled replicative polymerase is replaced with a specialized polymerase, while PCNA acetylation may reduce the processivity of replicative polymerases to promote homologous recombination-dependent repair. While regulatory functions of PCNA ubiquitination and acetylation have been well established, the regulation of PCNA-binding proteins remains underexplored. Considering the vast number of PCNA-binding proteins, many of which have similar PCNA binding affinities, the question arises as to the regulation of the strength and sequence of their binding to PCNA. Here I provide an overview of post-translational modifications on both PCNA and PCNA-interacting proteins and discuss their relevance for the regulation of the dynamic processes of DNA replication and repair.

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TRIM28 functions as the SUMO E3 ligase for PCNA in prevention of transcription induced DNA breaks

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.2004122117 ◽

2020 ◽

Vol 117 (38) ◽

pp. 23588-23596

Author(s):

Min Li ◽

Xiaohua Xu ◽

Chou-Wei Chang ◽

Yilun Liu

Keyword(s):

Rna Polymerase Ii ◽

Proliferating Cell Nuclear Antigen ◽

E3 Ligase ◽

Nuclear Antigen ◽

Dna Breaks ◽

Interacting Protein ◽

Proteomic Approach ◽

Sumo E3 Ligase ◽

Proliferating Cell

In human cells, the DNA replication factor proliferating cell nuclear antigen (PCNA) can be conjugated to either the small ubiquitinlike modifier SUMO1 or SUMO2, but only SUMO2-conjugated PCNA is induced by transcription to facilitate resolution of transcription–replication conflict (TRC). To date, the SUMO E3 ligase that provides substrate specificity for SUMO2-PCNA conjugation in response to TRC remains unknown. Using a proteomic approach, we identified TRIM28 as the E3 ligase that catalyzes SUMO2-PCNA conjugation. In vitro, TRIM28, together with the RNA polymerase II (RNAPII)-interacting protein RECQ5, promotes SUMO2-PCNA conjugation but inhibits SUMO1-PCNA formation. This activity requires a PCNA-interacting protein (PIP) motif located within the bromodomain of TRIM28. In cells, TRIM28 interaction with PCNA on human chromatin is dependent on both transcription and RECQ5, and SUMO2-PCNA level correlates with TRIM28 expression. As a consequence, TRIM28 depletion led to RNAPII accumulation at TRC sites, and expression of a TRIM28 PIP mutant failed to suppress TRC-induced DNA breaks.

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A Peptide Mimicking a Region in Proliferating Cell Nuclear Antigen Specific to Key Protein Interactions Is Cytotoxic to Breast Cancer

Molecular Pharmacology ◽

10.1124/mol.114.093211 ◽

2014 ◽

Vol 87 (2) ◽

pp. 263-276 ◽

Cited By ~ 20

Author(s):

Shanna J. Smith ◽

Long Gu ◽

Elizabeth A. Phipps ◽

Lacey E. Dobrolecki ◽

Karla S. Mabrey ◽

...

Keyword(s):

Breast Cancer ◽

Protein Interactions ◽

Proliferating Cell Nuclear Antigen ◽

Nuclear Antigen ◽

Proliferating Cell

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Structure of the processive human Pol δ holoenzyme

10.1101/872879 ◽

2019 ◽

Author(s):

Claudia Lancey ◽

Muhammad Tehseen ◽

Vlad-Stefan Raducanu ◽

Fahad Rashid ◽

Nekane Merino ◽

...

Keyword(s):

Dna Polymerase ◽

Proliferating Cell Nuclear Antigen ◽

Nuclear Antigen ◽

Structural Basis ◽

Catalytic Core ◽

Regulatory Subunits ◽

Functional Interactions ◽

Proliferating Cell ◽

Lagging Strand ◽

Catalytic Cleft

In eukaryotes, DNA polymerase δ (Pol δ) bound to the proliferating cell nuclear antigen (PCNA) replicates the lagging strand and cooperates with flap endonuclease 1 (FEN1) to process the Okazaki fragments for their ligation. We present the high-resolution cryo-EM structure of the human processive Pol δ-DNA-PCNA complex in the absence and presence of FEN1. Pol δ is anchored to one of the three PCNA monomers through the C-terminal domain of the catalytic subunit. The catalytic core sits on top of PCNA in an open configuration while the regulatory subunits project laterally. This arrangement allows PCNA to thread and stabilize the DNA exiting the catalytic cleft and recruit FEN1 to one unoccupied monomer in a toolbelt fashion. Alternative holoenzyme conformations reveal important functional interactions that maintain PCNA orientation during synthesis. This work sheds light on the structural basis of Pol δ’s activity in replicating the human genome.

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Structural Basis for Novel Interactions between Human Translesion Synthesis Polymerases and Proliferating Cell Nuclear Antigen

Journal of Biological Chemistry ◽

10.1074/jbc.m809745200 ◽

2009 ◽

Vol 284 (16) ◽

pp. 10552-10560 ◽

Cited By ~ 102

Author(s):

Asami Hishiki ◽

Hiroshi Hashimoto ◽

Tomo Hanafusa ◽

Keijiro Kamei ◽

Eiji Ohashi ◽

...

Keyword(s):

Proliferating Cell Nuclear Antigen ◽

Translesion Synthesis ◽

Nuclear Antigen ◽

Structural Basis ◽

Novel Interactions ◽

Proliferating Cell

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Identification of PCNA interacting protein motifs in human DNA polymerase delta

10.1101/2020.02.29.971473 ◽

2020 ◽

Author(s):

Prashant Khandagale ◽

Shweta Thakur ◽

Narottam Acharya

Keyword(s):

Dna Polymerase ◽

Proliferating Cell Nuclear Antigen ◽

Nuclear Antigen ◽

Dna Polymerase Delta ◽

Interacting Protein ◽

Protein Motifs ◽

Human Dna ◽

Biochemical Studies ◽

Processivity Factor ◽

Proliferating Cell

AbstractDNA polymerase delta (Polδ) is a highly processive essential replicative DNA polymerase. In humans, Polδ holoenzyme consists of p125, p50, p68, and p12 subunits and recently, we have shown that p12 exists as a dimer. Extensive biochemical studies suggest that all the subunits of Polδ interact with the processivity factor proliferating cell nuclear antigen (PCNA) to carry out a pivotal role in genomic DNA replication. While PCNA interaction protein (PIP) motifs in p68, p50 and p12 have been mapped, the PIP in p125, the catalytic subunit of the holoenzyme, remains elusive. Therefore, in this study by using multiple approaches we have conclusively mapped a non-canonical PIP box from residues 999VGGLLAFA1008 in p125, which binds to inter domain-connecting loop of PCNA with high affinity. Collectively, including previous studies, we conclude that similar to S. cerevisiae Polδ, each of the human Polδ subunits possess motif to interact with PCNA and significantly contribute towards the processive nature of this replicative DNA polymerase.

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A crystallization and preliminary X-ray diffraction study of the Arabidopsis thaliana proliferating cell nuclear antigen (PCNA2) alone and in a complex with a PIP-box peptide from Flap endonuclease 1

Acta Biochimica Polonica ◽

10.18388/abp.2020_2896 ◽

2020 ◽

Author(s):

Ewa Kowalska ◽

Wojciech Strzałka ◽

Takuji Oyama

Keyword(s):

Arabidopsis Thaliana ◽

Proliferating Cell Nuclear Antigen ◽

Nuclear Antigen ◽

X Ray Diffraction ◽

Interacting Protein ◽

Space Group ◽

X Ray ◽

Flap Endonuclease 1 ◽

Living Organisms ◽

Proliferating Cell

DNA replication is an important event for all living organisms and the mechanism is essentially conserved from archaea, bacteria to eukaryotes. Proliferating cell nuclear antigen (PCNA) acts as the universal platform for many DNA transacting proteins. Flap endonuclease 1 (FEN1) is one such enzyme whose activity is largely affected by the interaction with PCNA. To elucidate the key interactions between plant PCNA and FEN1 and possible structural change of PCNA caused by binding of FEN1 at the atomic level, crystallization and preliminary studies of X-ray diffraction of crystals of Arabidopsis thaliana PCNA2 (AtPCNA2) alone and in a complex with a peptide derived from AtFEN1, which contains a typical PCNA-interacting protein (PIP)-box motif, were performed. Both peptide-free and peptide-bound AtPCNA2s were crystallized using the same reservoir solution but in different crystal systems, indicating that the peptide affected the intermolecular interactions in the crystals. Crystals of AtPCNA2 belonged to the hexagonal space group P63, while those of the peptide-bound AtPCNA2 belonged to the rhombohedral space group H3, both of which could contain the functional homo-trimers.

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