Proliferating Cell Nuclear Antigen Subunit Research Articles

Identification of New Mutations at the PCNA Subunit Interface that Block Translesion Synthesis

Proliferating cell nuclear antigen (PCNA) plays an essential role in DNA replication and repair by interacting with a large number of proteins involved in these processes. Two amino acid substitutions in PCNA, both located at the subunit interface, have previously been shown to block translesion synthesis (TLS), a pathway for bypassing DNA damage during replication. To better understand the role of the subunit interface in TLS, we used random mutagenesis to generate a set of 33 PCNA mutants with substitutions at the subunit interface. We assayed the full set of mutants for viability and sensitivity to ultraviolet (UV) radiation. We then selected a subset of 17 mutants and measured their rates of cell growth, spontaneous mutagenesis, and UV-induced mutagenesis. All except three of these 17 mutants were partially or completely defective in induced mutagenesis, which indicates a partial or complete loss of TLS. These results demonstrate that the integrity of the subunit interface of PCNA is essential for efficient TLS and that even conservative substitutions have the potential to disrupt this process.

Interactome Analysis and Docking Sites of PCNA Subunits Reveal New Function in Arabidopsis thaliana

Proliferating Cell Nuclear Antigen (PCNA) is a protein known for its function in DNA replication in all eukaryotic cells. PCNA also has a role in DNA repair when exterior factors, like mutagenic UV-B radiation of sunlight, free radicals, ionizing radiations, chemical mutagens, etc., result in damage to DNA either genetoxically or cytotoxically. PCNA is usually of a toroidal shape and is made-up of either homo- or hetero-trimers. PCNA in Arabidopsis thaliana has two subunits; PCNA1 and PCNA2 that forms a homotrimeric structure. We found that the two subunits (PCNA1 and PCNA2) have a 97% similarity in their structures. Many studies have been conducted on PCNA from humans or yeast, but there is very little research done on plant PCNA. This study aims to close that gap, by conducting a study on PCNA subunits in Arabidopsis thaliana by predicting its 3D structure, domain analysis, subcellular localization, docking site prediction and PCNA interactome. Interactome analysis of PCNA revealed that it interacts with many proteins in DNA repair, such as Rad5, RAD9, RFC3, RFC5, RFC1, which are all a part of pathways involved in DNA repair. Interestingly we found that PCNA interacts with AtBRD1which is controlling shoot differentiation and meristem organization. Keywords: Arabidopsis thaliana, 3-D Structure, Interactome, docking sites, DNA Repair, PCNA.

Electron donation to an archaeal cytochrome P450 is enhanced by PCNA‐mediated selective complex formation with foreign redox proteins

Cytochrome P450 monooxygenases (P450s) are environmentally friendly biocatalysts that catalyze diverse chemical reactions using molecular oxygen under mild reaction conditions. P450s are activated upon receiving electrons from specific redox partner proteins, although the redox partners for most bacterial/archaeal P450s are not yet identified. Thus, it is important to establish a variety of efficient and versatile electron transfer systems from NAD(P)H to P450s for the design of biocatalysts. Sulfolobus solfataricus possesses a heterotrimeric proliferating cell nuclear antigen (PCNA). Fusion of the PCNA subunits to S. acidocaldarius P450 (CYP119) and the Pseudomonas putida redox proteins, putidaredoxin (PdX) and putidaredoxin reductase (PdR), yielded a selective protein complex containing one molecule each of the three proteins. The PCNA-mediated heterotrimerization of CYP119, PdX, and PdR enhanced the CYP119 activity, likely as a result of high local concentrations of the two redox proteins toward CYP119. Therefore, the PCNA-mediated formation of the complex containing PdX and PdR might be applicable for harnessing the utility of P450s whose redox partners are not yet identified.

Fine Tuning of Spatial Arrangement of Enzymes in a PCNA-Mediated Multienzyme Complex Using a Rigid Poly-L-Proline Linker

Inspired by natural multienzyme complexes, many types of artificial multienzyme complexes have recently been constructed. We previously constructed a self-assembled complex of a bacterial cytochrome P450 and its ferredoxin and ferredoxin reductase partners using heterotrimerization of proliferating cell nuclear antigen (PCNA) from Sulfolobus solfataricus. In this study, we inserted different peptide linkers between ferredoxin and the PCNA subunit, and examined the effect on activity of the self-assembled multienzyme complex. Although the activity was affected by the lengths of both the rigid poly-L-proline-rich linkers and the flexible Gly4-Ser repeating linkers, the poly-L-proline-rich linkers provided the greatest activity enhancement. The optimized poly-L-proline-rich linker enhanced the activity 1.9-fold compared with the GGGGSLVPRGSGGGGS linker used in the previously reported complex, while the Gly4-Ser repeating linkers, (G4S)n (n = 1–6), did not yield higher activity than the maximum activity by the optimized poly-L-proline linker. Both the rigidity/flexibility and length of the peptide linker were found to be important for enhancing the overall activity of the multienzyme complex.

Distinct Structural Alterations in Proliferating Cell Nuclear Antigen Block DNA Mismatch Repair

During DNA replication, mismatches and small loops in the DNA resulting from insertions or deletions are repaired by the mismatch repair (MMR) machinery. Proliferating cell nuclear antigen (PCNA) plays an important role in both mismatch-recognition and resynthesis stages of MMR. Previously, two mutant forms of PCNA were identified that cause defects in MMR with little, if any, other defects. The C22Y mutant PCNA protein completely blocks MutSα-dependent MMR, and the C81R mutant PCNA protein partially blocks both MutSα-dependent and MutSβ-dependent MMR. In order to understand the structural and mechanistic basis by which these two amino acid substitutions in PCNA proteins block MMR, we solved the X-ray crystal structures of both mutant proteins and carried out further biochemical studies. We found that these amino acid substitutions lead to subtle, distinct structural changes in PCNA. The C22Y substitution alters the positions of the α-helices lining the central hole of the PCNA ring, whereas the C81R substitution creates a distortion in an extended loop near the PCNA subunit interface. We conclude that the structural integrity of the α-helices lining the central hole and this loop are both necessary to form productive complexes with MutSα and mismatch-containing DNA.

Introduction of selective intersubunit disulfide bonds into self‐assembly protein scaffold to enhance an artificial multienzyme complex's activity

In nature, many enzymes participating in multienzyme reactions are often assembled to enhance efficiencies of multiple reactions. Therefore, much attention has been focused on self-assembly of multiple enzymes fused with a protein/peptide that interacts with a specific protein to enhance artificial multienzyme reactions. Sulfolobus solfataricus proliferating cell nuclear antigen (PCNA) is a ring-shaped symmetric heterotrimer consisting of PCNA1, PCNA2 and PCNA3. Multiple enzymes can be co-localized on the PCNA ring by fusing them to the C-termini of the three PCNA subunits. However, an advantage of the specific non-covalent complex formation is inextricably associated with the disadvantage of its concentration-dependent dissociation. In this study, disulfide bonds were introduced between the PCNA subunits by Cys substitution at the sites neighboring the interface for heterotrimerization. Selective intersubunit disulfide bond formation between PCNA1 and PCNA3 and between PCNA2 and PCNA3 by a natural oxidizing reagent successfully stabilized an artificial multienzyme complex, which is composed of a bacterial cytochrome P450 and its two redox partner proteins. The covalent stabilization of the multienzyme complex enhanced its cytochrome P450 activity because of the absence of inactive dissociated components.

Architecture of the DNA polymerase B-proliferating cell nuclear antigen (PCNA)-DNA ternary complex

DNA replication in archaea and eukaryotes is executed by family B DNA polymerases, which exhibit full activity when complexed with the DNA clamp, proliferating cell nuclear antigen (PCNA). This replication enzyme consists of the polymerase and exonuclease moieties responsible for DNA synthesis and editing (proofreading), respectively. Because of the editing activity, this enzyme ensures the high fidelity of DNA replication. However, it remains unclear how the PCNA-complexed enzyme temporally switches between the polymerizing and editing modes. Here, we present the three-dimensional structure of the Pyrococcus furiosus DNA polymerase B-PCNA-DNA ternary complex, which is the core component of the replisome, determined by single particle electron microscopy of negatively stained samples. This structural view, representing the complex in the editing mode, revealed the whole domain configuration of the trimeric PCNA ring and the DNA polymerase, including protein-protein and protein-DNA contacts. Notably, besides the authentic DNA polymerase-PCNA interaction through a PCNA-interacting protein (PIP) box, a novel contact was found between DNA polymerase and the PCNA subunit adjacent to that with the PIP contact. This contact appears to be responsible for the configuration of the complex specific for the editing mode. The DNA was located almost at the center of PCNA and exhibited a substantial and particular tilt angle against the PCNA ring plane. The obtained molecular architecture of the complex, including the new contact found in this work, provides clearer insights into the switching mechanism between the two distinct modes, thus highlighting the functional significance of PCNA in the replication process.

Multiscale Modeling of PCNA - Ubiquitin Interactions

Covalent attachment of ubiquitin (Ub) to proliferating cell nuclear antigen (PCNA) plays a crucial role in translesion synthesis (TLS). However, structural knowledge of the complex between Ub and PCNA is currently lacking. The problem is important from a biological perspective since ubiquitinated PCNA is involved in the recruitment of specialized lesion bypass polymerases. A loss of regulation of TLS and other damage-avoidance pathways can lead to a variety of cell fates including apoptosis and uncontrolled cell growth. We have modeled the Ub-PCNA complex using a combination of tethered Brownian dynamics (TBD), protein-protein docking with RosettaDock, flexible loop modeling with ModLoop, and molecular dynamics(MD). The TBD simulations were used to generate a large ensemble of electrostatically and geometrically favorable configurations, subsequently used as a starting point for local docking searches. The final models were refined with all-atom explicit solvent MD simulations. Ubiquitin was found to bind in a groove on the PCNA surface directly above the PCNA subunit interface. A mutation originally identified in genetics screens (pol30-113) and known to interfere with TLS, is positioned directly beneath the bound ubiquitin in our models. Thus, the results provide unexpected insight into previously unexplained biological observations.View Large Image | View Hi-Res Image | Download PowerPoint Slide

Spatial subunit distribution and in vitro functions of the novel trimeric PCNA complex from Sulfolobus tokodaii

The relationships among three PCNA (proliferating cell nuclear antigen) subunits in the hyperthermophilic archaeon Sulfolobus tokodaii (StoPCNAs) were analyzed and the effects of two PCNA complexes on the activities of the DNA helicase Hjm, DNA Ligase I, and Holliday junction specific endonuclease Hjc were tested. There was no strong self-interaction of each StoPCNA. StoPCNA1 and StoPCNA3 interacted with each other, so did StoPCNA2 and StoPCNA3, but no interaction between StoPCNA1 and StoPCNA2 was observed. Two trimeric complexes (designed StoPCNA123 and StoPCNA323) were formed in vitro and it was determined that StoPCNA323 was composed of one StoPCNA2 and two StoPCNA3 subunits, with StoPCNA2 bridging the two StoPCNA3 subunits. Both complexes inhibited the unwinding activity of Hjm and the ligation activity of DNA Ligase I. In contrast, both stimulated the Holliday junction cleavage activity of Hjc. Our results provide further evidence that in crenarchaea, the PCNAs exhibit diversity in subunit interaction and complex formation.

Architecture and Assembly of Poly-SUMO Chains on PCNA in Saccharomyces cerevisiae

Posttranslational modifications of proliferating cell nuclear antigen (PCNA), the eukaryotic processivity clamp for DNA polymerases, regulate the pathways by which replication problems are resolved. In the budding yeast Saccharomyces cerevisiae, ubiquitylation of PCNA in response to DNA damage facilitates the replicative bypass of lesions, whereas conjugation of the ubiquitin-related modifier (SUMO) prevents unscheduled crossover events during S phase. We have analyzed the SUMO modification pattern of budding yeast PCNA in vivo and in vitro and found that most aspects of our in vitro sumoylation reactions reflect the situation under physiological conditions. We show that two oligomeric SUMO chains of two to three moieties each, linked via internal sumoylation consensus motifs within the SUMO sequence, are assembled on PCNA. The SUMO-specific ligase Siz1 both stimulates the overall efficiency of sumoylation and selects the modification site on PCNA. Furthermore, ubiquitin and SUMO chains are assembled independently, and we found evidence that both modifiers can coexist in vivo on a common PCNA subunit. Our results demonstrate for the first time the in vivo assembly of polymeric SUMO chains of defined linkage on a physiological substrate in yeast, but they also indicate that SUMO–SUMO polymers are dispensable for PCNA SUMO function in replication and recombination.

Reactive oxygen‐mediated damage to a human DNA replication and repair protein

Ultraviolet A (UVA) makes up more than 90% of incident terrestrial ultraviolet radiation. Unlike shorter wavelength UVB, which damages DNA directly, UVA is absorbed poorly by DNA and is therefore considered to be less hazardous. Organ transplant patients treated with the immunosuppressant azathioprine frequently develop skin cancer. Their DNA contains 6-thioguanine-a base analogue that generates DNA-damaging singlet oxygen ((1)O(2)) when exposed to UVA. Here, we show that this (1)O(2) damages proliferating cell nuclear antigen (PCNA), the homotrimeric DNA polymerase sliding clamp. It causes covalent oxidative crosslinking between the PCNA subunits through a histidine residue in the intersubunit domain. Crosslinking also occurs after treatment with higher-although still moderate-doses of UVA alone or with chemical oxidants. Chronic accumulation of oxidized proteins is linked to neurodegenerative disorders and ageing. Our findings identify oxidative damage to an important DNA replication and repair protein as a previously unrecognized hazard of acute oxidative stress.

A single amino acid substitution in the DNA-binding domain of Aeropyrum pernix DNA ligase impairs its interaction with proliferating cell nuclear antigen

Proliferating cell nuclear antigen (PCNA) is known as a DNA sliding clamp that acts as a platform for the assembly of enzymes involved in DNA replication and repair. Previously, it was reported that a crenarchaeal PCNA formed a heterotrimeric structure, and that each PCNA subunit has distinct binding specificity to PCNA-binding proteins. Here we describe the PCNA-binding properties of a DNA ligase from the hyperthermophilic crenarchaeon Aeropyrum pernix K1. Based on our findings on the Pyrococcus furiosus DNA ligase-PCNA interaction, we predicted that the aromatic residue, Phe132, in the DNA-binding domain of A. pernix DNA ligase (ApeLig) would play a critical role in binding to A. pernix PCNA (ApePCNA). Surface plasmon resonance analyses revealed that the ApeLig F132A mutant does not interact with an immobilized subunit of ApePCNA. Furthermore, we could not detect any stimulation of the ligation activity of the ApeLig F132A protein by ApePCNA in vitro. These results indicated that the phenylalanine, which is located in our predicted PCNA-binding region in ApeLig, has a critical role for the physical and functional interaction with ApePCNA.

Proliferating cell nuclear antigen loaded onto double-stranded DNA: dynamics, minor groove interactions and functional implications

Proliferating cell nuclear antigen (PCNA) acts as a biologically essential processivity factor that encircles DNA and provides binding sites for polymerase, flap endonuclease-1 (FEN-1) and ligase during DNA replication and repair. We have computationally characterized the interactions of human and Archaeoglobus fulgidus PCNA trimer with double-stranded DNA (ds DNA) using multi-nanosecond classical molecular dynamics simulations. The results reveal the interactions of DNA passing through the PCNA trimeric ring including the contacts formed, overall orientation and motion with respect to the sliding clamp. Notably, we observe pronounced tilting of the axis of dsDNA with respect to the PCNA ring plane reflecting interactions between the DNA phosphodiester backbone and positively charged arginine and lysine residues lining the PCNA inner surface. Covariance matrix analysis revealed a pattern of correlated motions within and between the three equivalent subunits involving the PCNA C-terminal region and linker strand associated with partner protein binding sites. Additionally, principal component analysis identified low frequency global PCNA subunit motions suitable for translocation along duplex DNA. The PCNA motions and interactions with the DNA minor groove, identified here computationally, provide an unexpected basis for PCNA to act in the coordinated handoff of intermediates from polymerase to FEN-1 to ligase during DNA replication and repair.

A Flexible Interface between DNA Ligase and PCNA Supports Conformational Switching and Efficient Ligation of DNA

DNA sliding clamps encircle DNA and provide binding sites for many DNA-processing enzymes. However, it is largely unknown how sliding clamps like proliferating cell nuclear antigen (PCNA) coordinate multistep DNA transactions. We have determined structures of Sulfolobus solfataricus DNA ligase and heterotrimeric PCNA separately by X-ray diffraction and in complex by small-angle X-ray scattering (SAXS). Three distinct PCNA subunits assemble into a protein ring resembling the homotrimeric PCNA of humans but with three unique protein-binding sites. In the absence of nicked DNA, the Sulfolobus solfataricus DNA ligase has an open, extended conformation. When complexed with heterotrimeric PCNA, the DNA ligase binds to the PCNA3 subunit and ligase retains an open, extended conformation. A closed, ring-shaped conformation of ligase catalyzes a DNA end-joining reaction that is strongly stimulated by PCNA. This open-to-closed switch in the conformation of DNA ligase is accommodated by a malleable interface with PCNA that serves as an efficient platform for DNA ligation.

Structure of an archaeal PCNA1–PCNA2–FEN1 complex: elucidating PCNA subunit and client enzyme specificity

The archaeal/eukaryotic proliferating cell nuclear antigen (PCNA) toroidal clamp interacts with a host of DNA modifying enzymes, providing a stable anchorage and enhancing their respective processivities. Given the broad range of enzymes with which PCNA has been shown to interact, relatively little is known about the mode of assembly of functionally meaningful combinations of enzymes on the PCNA clamp. We have determined the X-ray crystal structure of the Sulfolobus solfataricus PCNA1–PCNA2 heterodimer, bound to a single copy of the flap endonuclease FEN1 at 2.9 Å resolution. We demonstrate the specificity of interaction of the PCNA subunits to form the PCNA1–PCNA2–PCNA3 heterotrimer, as well as providing a rationale for the specific interaction of the C-terminal PIP-box motif of FEN1 for the PCNA1 subunit. The structure explains the specificity of the individual archaeal PCNA subunits for selected repair enzyme ‘clients’, and provides insights into the co-ordinated assembly of sequential enzymatic steps in PCNA-scaffolded DNA repair cascades.