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

Biasio, Alfredo De; Opakua, Alain Ibáñez de; Mortuza, Gulnahar B.; Molina, Rafael; Cordeiro, Tiago N.; Castillo, Francisco; Villate, Maider; Merino, Nekane; Delgado, Sandra; Gil, David; Luque, Irene; Diercks, Tammo; Bernadó, Pau; Montoya, Guillermo; Blanco, Francisco J.

doi:10.1038/ncomms7439

Cited by 71 publications

(124 citation statements)

References 60 publications

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“…Therefore, the proposed sliding mechanism of PCNA may as well be important for the assembly of functional complexes that involve TLS polymerases. Interestingly, the binding site of DNA on the inner wall of the PCNA ring partly overlaps with that of p15 PAF , an intrinsically disordered protein that regulates TLS via its interaction with PCNA212223. Binding of p15 PAF could modulate the PCNA sliding mechanism and dynamics, which may play a role in DNA repair.…”

Section: Discussionmentioning

confidence: 99%

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Structural basis of human PCNA sliding on DNA

et al. 2017

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Sliding clamps encircle DNA and tether polymerases and other factors to the genomic template. However, the molecular mechanism of clamp sliding on DNA is unknown. Using crystallography, NMR and molecular dynamics simulations, here we show that the human clamp PCNA recognizes DNA through a double patch of basic residues within the ring channel, arranged in a right-hand spiral that matches the pitch of B-DNA. We propose that PCNA slides by tracking the DNA backbone via a ‘cogwheel' mechanism based on short-lived polar interactions, which keep the orientation of the clamp invariant relative to DNA. Mutation of residues at the PCNA–DNA interface has been shown to impair the initiation of DNA synthesis by polymerase δ (pol δ). Therefore, our findings suggest that a clamp correctly oriented on DNA is necessary for the assembly of a replication-competent PCNA-pol δ holoenzyme.

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Section: Discussionmentioning

confidence: 99%

“…The purified protein contained the extra sequence GPH- at the N terminus. The PCNA sample for crystallization was obtained by introducing two additional purification steps23. The sample cleaved with PreScission protease was dialysed against 50 mM sodium acetate pH 5.5, 100 mM NaCl.…”

Section: Methodsmentioning

confidence: 99%

Structural basis of human PCNA sliding on DNA

et al. 2017

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“…At the same time, SPARTAN plays a crucial role in switching off TLS, by recruiting the ubiquitin-selective chaperone p97 to remove Polη from DNA (Davis et al, 2012; Mosbech et al, 2012). The small PCNA interacting protein PAF15 also participates in turning off TLS by tightly associating with PCNA following lesion bypass, out-competing TLS polymerases (De Biasio et al, 2015; Povlsen et al, 2012). …”

Section: Translesion Synthesismentioning

confidence: 99%

Forging Ahead through Darkness: PCNA, Still the Principal Conductor at the Replication Fork

2017

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Proliferating Cell Nuclear Antigen (PCNA) lies at the center of the faithful duplication of eukaryotic genomes. With its distinctive doughnut-shaped molecular structure, PCNA was originally studied for its role in stimulating DNA polymerases. However, we now know that PCNA does much more than promote processive DNA synthesis. Because of the complexity of the events involved, cellular DNA replication poses major threats to genomic integrity. Whatever predicament lies ahead for the replication fork, PCNA is there to orchestrate the events necessary to handle it. Through its many protein interactions and various post-translational modifications, PCNA has far-reaching impacts on a myriad of cellular functions.

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“…The structural details of the interaction of PCNA with several proteins have been revealed by crystallography. The crystallographic structures available correspond to PIP-box peptides derived from seven IDPs or IDRs, namely p21 (22), Polδ p66 (23), FEN1 (24), RNaseH2B (25), Polη (26), Polκ (26) and p15 (27). PIP-boxes display similar structural characteristics in the bound state, featuring a short 3 10 helix but diverging in the structural content and the orientation of the flanking regions (23,27).…”

Section: Introductionmentioning

confidence: 99%

Disentangling polydispersity in the PCNA−p15PAF complex, a disordered, transient and multivalent macromolecular assembly

Cordeiro

Chen

Biasio

et al. 2016

Nucleic Acids Research

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The intrinsically disordered p15PAF regulates DNA replication and repair when interacting with the Proliferating Cell Nuclear Antigen (PCNA) sliding clamp. As many interactions between disordered proteins and globular partners involved in signaling and regulation, the complex between p15PAF and trimeric PCNA is of low affinity, forming a transient complex that is difficult to characterize at a structural level due to its inherent polydispersity. We have determined the structure, conformational fluctuations, and relative population of the five species that coexist in solution by combining small-angle X-ray scattering (SAXS) with molecular modelling. By using explicit ensemble descriptions for the individual species, built using integrative approaches and molecular dynamics (MD) simulations, we collectively interpreted multiple SAXS profiles as population-weighted thermodynamic mixtures. The analysis demonstrates that the N-terminus of p15PAF penetrates the PCNA ring and emerges on the back face. This observation substantiates the role of p15PAF as a drag regulating PCNA processivity during DNA repair. Our study reveals the power of ensemble-based approaches to decode structural, dynamic, and thermodynamic information from SAXS data. This strategy paves the way for deciphering the structural bases of flexible, transient and multivalent macromolecular assemblies involved in pivotal biological processes.

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

Cited by 71 publications

References 60 publications

Structural basis of human PCNA sliding on DNA

Structural basis of human PCNA sliding on DNA

Forging Ahead through Darkness: PCNA, Still the Principal Conductor at the Replication Fork

Disentangling polydispersity in the PCNA−p15PAF complex, a disordered, transient and multivalent macromolecular assembly

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