Explore the vast range of titles available.

• Background PCNA (proliferating cell nuclear antigen) has been found in the nuclei of yeast, plant and animal cells that undergo cell division, suggesting a function in cell cycle regulation and/or DNA replication. It subsequently became clear that PCNA also played a role in other processes involving the cell genome. • Scope This review discusses eukaryotic PCNA, with an emphasis on plant PCNA, in terms of the protein structure and its biochemical properties as well as gene structure, organization, expression and function. PCNA exerts a tripartite function by operating as (1) a sliding clamp during DNA synthesis, (2) a polymerase switch factor and (3) a recruitment factor. Most of its functions are mediated by its interactions with various proteins involved in DNA synthesis, repair and recombination as well as in regulation of the cell cycle and chromatid cohesion. Moreover, post-translational modifications of PCNA play a key role in regulation of its functions. Finally, a phylogenetic comparison of PCNA genes suggests that the multi-functionality observed in most species is a product of evolution. • Conclusions Most plant PCNAs exhibit features similar to those found for PCNAs of other eukaryotes. Similarities include: (1) a trimeric ring structure of the PCNA sliding clamp, (2) the involvement of PCNA in DNA replication and repair, (3) the ability to stimulate the activity of DNA polymerase δ and (4) the ability to interact with p21, a regulator of the cell cycle. However, many plant genomes seem to contain the second, probably functional, copy of the PCNA gene, in contrast to PCNA pseudogenes that are found in mammalian genomes.

DNA damage tolerance (DDT) is an important pathway that allows cells to bypass DNA lesions during replication. DDT is orchestrated by ubiquitination of PCNA, where monoubiquitinated PCNA (PCNA-Ub) initiates recruitment of TLS polymerases but also serves as a substrate for further ubiquitination, forming K63-polyubiquitinated PCNA that leads to HR-mediated bypass mechanisms. Recent work on USP1/UAF1 inhibition revealed that formation of K48-linked chains also occurs on PCNA, resulting in its proteasomal degradation. USP1/UAF1 is established as deubiquitinating enzyme (DUB) for PCNA-Ub, but little is known about removal of ubiquitin chains on PCNA. Here we show that USP1/UAF1 cleaves both K48 and K63-linked ubiquitin chains on PCNA efficiently, using an exo-cleavage mechanism. Kinetic analysis reveals that USP1/UAF1 prefers cleaving the ubiquitin-ubiquitin bond over cleavage of the ubiquitin-PCNA bond and therefore treats poly- and monoubiquitinated PCNA as different substrates. A cryo-EM structure of USP1/UAF1 with a K63-diubiquitin and structure-based mutagenesis suggests that this mechanistic preference is maintained in evolution. This unusual mechanism can cause temporal enrichment of monoubiquitinated PCNA during polyubiquitination. It will be interesting to see how this affects DDT pathway balance. DNA damage tolerance is regulated by ubiquitination of PCNA. Here, the authors present kinetic and structural studies showing that USP1/UAF1 prefers trimming K63- and K48-ubiquitin chains down over cleavage of monoubiquitinated PCNA. Mutant analysis suggests evolutionary preservation of this mechanism.

DNA replication requires the sliding clamp, a ring-shaped protein complex that encircles DNA, where it acts as an essential cofactor for DNA polymerases and other proteins. The sliding clamp needs to be opened and installed onto DNA by a clamp loader ATPase of the AAA+ family. The human clamp loader replication factor C (RFC) and sliding clamp proliferating cell nuclear antigen (PCNA) are both essential and play critical roles in several diseases. Despite decades of study, no structure of human RFC has been resolved. Here, we report the structure of human RFC bound to PCNA by cryogenic electron microscopy to an overall resolution of ∼3.4 Å. The active sites of RFC are fully bound to adenosine 5′-triphosphate (ATP) analogs, which is expected to induce opening of the sliding clamp. However, we observe the complex in a conformation before PCNA opening, with the clamp loader ATPase modules forming an overtwisted spiral that is incapable of binding DNA or hydrolyzing ATP. The autoinhibited conformation observed here has many similarities to a previous yeast RFC:PCNA crystal structure, suggesting that eukaryotic clamp loaders adopt a similar autoinhibited state early on in clamp loading. Our results point to a “limited change/induced fit” mechanism in which the clamp first opens, followed by DNA binding, inducing opening of the loader to release autoinhibition. The proposed change from an overtwisted to an active conformation reveals an additional regulatory mechanism for AAA+ ATPases. Finally, our structural analysis of disease mutations leads to a mechanistic explanation for the role of RFC in human health.

FAN1 is a DNA dependent nuclease whose proper function is essential for maintaining human health. For example, a genetic variant in FAN1, Arg507 to His hastens onset of Huntington’s disease, a repeat expansion disorder for which there is no cure. How the Arg507His mutation affects FAN1 structure and enzymatic function is unknown. Using cryo-EM and biochemistry, we have discovered that FAN1 arginine 507 is critical for its interaction with PCNA, and mutation of Arg507 to His attenuates assembly of the FAN1–PCNA complex on a disease-relevant extrahelical DNA extrusions formed within DNA repeats. This mutation concomitantly abolishes PCNA–FAN1–dependent cleavage of such extrusions, thus unraveling the molecular basis for a specific mutation in FAN1 that dramatically hastens the onset of Huntington’s disease. These results underscore the importance of PCNA to the genome stabilizing function of FAN1. FAN1 nuclease removes DNA triplet repeat loops by a process that requires PCNA. Using cryo-EM, the authors elucidate this mechanism, and show that a Huntington’s disease modifying R507H mutation inactivates FAN1 by compromising the FAN1-PCNA complex.

FAN1 is an endo- and exo-nuclease involved in DNA and interstrand crosslink repair. Genome-wide association studies of people with Huntington’s disease revealed a strong association between the FAN1 R507H mutation and early disease onset, however the underlying mechanism(s) remains unclear. FAN1 has previously been implicated in modulating triplet repeat expansion in a PCNA dependent manner. To examine the role of PCNA on FAN1 activation, we solved the cryo-EM structures of a PCNA–FAN1–DNA complex. Our findings reveal that the FAN1 R507 residue directly interacts with PCNA D232. Biophysical interaction studies demonstrated that FAN1 enhances the binding affinity of PCNA for DNA, a synergistic effect disrupted in mutants carrying the R507H mutation. In contrast, PCNA does not affect the affinity of FAN1 for DNA but does modulate FAN1 activity upon ternary complex formation. The weakened and functionally altered FAN1 R507H–PCNA–DNA complex may partly impair the FAN1-mediated repair of CAG extrahelical extrusions, providing a potential explanation for the mutation’s role in accelerating disease progression. Patients with Huntington’s disease carrying the FAN1 R507H mutation have earlier than predicted onset of motoric symptoms. This study provides mechanistic insight into the interactions that may promote CAG repeat expansion. FAN1 R507 interacts with PCNA D232 and this interaction is impaired for FAN1 R507H, resulting in reduced FAN1 activity.

Strategies to resolve replication blocks are critical for the maintenance of genome stability. Among the factors implicated in the replication stress response is the ATP-dependent endonuclease ZRANB3. Here, we present the structure of the ZRANB3 HNH (His-Asn-His) endonuclease domain and provide a detailed analysis of its activity. We further define PCNA as a key regulator of ZRANB3 function, which recruits ZRANB3 to stalled replication forks and stimulates its endonuclease activity. Finally, we present the co-crystal structures of PCNA with two specific motifs in ZRANB3: the PIP box and the APIM motif. Our data provide important structural insights into the PCNA-APIM interaction, and reveal unexpected similarities between the PIP box and the APIM motif. We propose that PCNA and ATP-dependency serve as a multi-layered regulatory mechanism that modulates ZRANB3 activity at replication forks. Importantly, our findings allow us to interpret the functional significance of cancer associated ZRANB3 mutations. ZRANB3 (Zinc-finger, RAN-Binding domain containing 3) is a structure-specific endonuclease that is recruited to DNA breaks and stressed replication forks. Here the authors present the crystal structure of the ZRANB3 endonuclease domain and analyse how ZRANB3 is regulated by the DNA clamp PCNA.

Numerous human disorders, including Cockayne syndrome, UV-sensitive syndrome, xeroderma pigmentosum, and trichothiodystrophy, result from the mutation of genes encoding molecules important for nucleotide excision repair. Here, we describe a syndrome in which the cardinal clinical features include short stature, hearing loss, premature aging, telangiectasia, neurodegeneration, and photosensitivity, resulting from a homozygous missense (p.Ser228Ile) sequence alteration of the proliferating cell nuclear antigen (PCNA). PCNA is a highly conserved sliding clamp protein essential for DNA replication and repair. Due to this fundamental role, mutations in PCNA that profoundly impair protein function would be incompatible with life. Interestingly, while the p.Ser228Ile alteration appeared to have no effect on protein levels or DNA replication, patient cells exhibited marked abnormalities in response to UV irradiation, displaying substantial reductions in both UV survival and RNA synthesis recovery. The p.Ser228Ile change also profoundly altered PCNA's interaction with Flap endonuclease 1 and DNA Ligase 1, DNA metabolism enzymes. Together, our findings detail a mutation of PCNA in humans associated with a neurodegenerative phenotype, displaying clinical and molecular features common to other DNA repair disorders, which we showed to be attributable to a hypomorphic amino acid alteration.

Proliferating cell nuclear antigen (PCNA) is an essential factor for DNA metabolism. The influence of PCNA on DNA replication and repair, combined with the high expression rate of PCNA in various tumours renders PCNA a promising target for cancer therapy. In this context, an autodisplay‐based screening method was developed to identify peptidic PCNA interaction inhibitors. A 12‐mer randomized peptide library consisting of 2.54 × 106 colony‐forming units was constructed and displayed at the surface of Escherichia coli BL21 (DE3) cells by autodisplay. Cells exhibiting an enhanced binding to fluorescent mScarlet‐I‐PCNA were enriched in four sorting rounds by flow cytometry. This led to the discovery of five peptide variants with affinity to mScarlet‐I‐PCNA. Among these, P3 (TCPLRWITHDHP) exhibited the highest binding signal. Subsequent flow cytometric analysis revealed a dissociation constant of 0.62 μM for PCNA‐P3 interaction. Furthermore, the inhibition of PCNA interactions was investigated using p15, a PIP‐box containing protein involved in DNA replication and repair. P3 inhibited the PCNA‐p1551‐70 interaction with a half maximal inhibitory activity of 16.2 μM, characterizing P3 as a potent inhibitor of the PCNA‐p15 interaction. Peptide surface display library screening was applied to identify new inhibitors of human PCNA/15 interaction, which plays a role in DNA replication. Five new inhibiting peptides were identified and the KD value of most potent P3 was determined.

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. Pol δ bound to the proliferating cell nuclear antigen (PCNA) replicates the lagging strand in eukaryotes and cooperates with flap endonuclease 1 (FEN1) to process the Okazaki fragments for their ligation. Here, the authors present a Cryo-EM structure of the human 4-subunit Pol δ bound to DNA and PCNA in a replicating state with an incoming nucleotide in the active site.

Proliferating cell nuclear antigen (PCNA) is an essential factor in DNA replication and repair. It forms a homotrimeric ring that embraces the DNA and slides along it, anchoring DNA polymerases and other DNA editing enzymes. It also interacts with regulatory proteins through a sequence motif known as PCNA Interacting Protein box (PIP-box). We here review the latest contributions to knowledge regarding the structure-function relationships in human PCNA, particularly the mechanism of sliding, and of the molecular recognition of canonical and non-canonical PIP motifs. The unique binding mode of the oncogene p15 is described in detail, and the implications of the recently discovered structure of PCNA bound to polymerase δ are discussed. The study of the post-translational modifications of PCNA and its partners may yield therapeutic opportunities in cancer treatment, in addition to illuminating the way PCNA coordinates the dynamic exchange of its many partners in DNA replication and repair.