Encyclopedia of Signaling Molecules

2018 Edition
| Editors: Sangdun Choi

Proliferating Cell Nuclear Antigen

  • Rashmi Maruti Hosalkar
  • Niharika Swain
  • Jayesh S. Khivasara
  • Samapika Routray
Reference work entry
DOI: https://doi.org/10.1007/978-3-319-67199-4_102006

Synonyms

Historical Background

Proliferation cell nuclear antigen (PCNA) was identified by two separate groups of researchers performing separate studies. Initially it was identified in a study on systemic lupus erythematous patients as protein present in the nucleus of dividing cells. During the same period, in another study, it was observed as a protein synthesized during S-phase of cell cycle and was termed as cyclin. However, further researches demonstrated that the proteins discovered in the above two studies were same and now are known by PCNA (Strzalka and Ziemienowicz 2011).

Gene Transcription

In 1989, Travali and coworkers molecularly cloned and sequenced human PCNA gene (Travali et al. 1989). It was sequenced from BamHi restriction site located 2.8 kb upstream from the site for transcription initiation (CAP site). It consists of six exons separated by five introns. Human PCNA gene has features that are noticeable such as absence of TATAA box on 5′flanking sequence, inverted CCAAT box at −95, direct CCAAT box at −141, presence of octamer ATTTGCT along with AP 1to4 and SP1 binding sites, E1A, and steroid response elements. The promoter region has six binding sites for polyoma T antigen (Baserga 1991). The human PCNA gene is localized on chromosome 20 and has four pseudogene PCNAP1, PCNAP2, LOC392454, and LOC390102 (Stoimenov and Lagerqvist 2012). In studies conducted, PCNA gene could be induced by varied growth factors such as platelet-derived growth factor, fibroblast growth factor, and epidermal growth factor. PCNA can also be induced by interleukin 2 and E1A protein of adenovirus (Travali et al. 1989).

Posttranslational Modifications

In vast majority of PCNA, some may undergo posttranslational modifications such as ubiquitylation, phosphorylation, acetylation, methylation, and sumoylation that affect its function (Fig. 1).
Proliferating Cell Nuclear Antigen, Fig. 1

Posttranscriptional modification of PCNA. Targets sites on PCNA (blue box). Enzymes acting on target sites (orange box). Posttranscriptional modification type (red box). Functions of PCNA after modification (green box)

Structure and Distribution

PCNA is a homotrimeric, acidic ring consisting of three identical PCNA monomers forming a sixfold symmetry joined in head to tail arrangement with an inner and outer diameter of 34 Å and 80 Å, respectively. Thus, the three interfaces form an inner ring enclosing 34 Å forming deoxyribose nucleic acid (DNA) and acting as a structural platform for DNA replication. PCNA monomer consists of two globular domains each containing two α-helices (positively charged) which line the central cavity and are perpendicular to DNA. These α-helices are supported by a continuous layer of nine β-sheet structures (negatively charged) extending across the interdomain boundary. The two domains are linked by a long flexible linker, interdomain-connecting loop (IDCL). One monomer interacts with another through β-sheet extension, mediated by a β-dimer interface that comprises extensive hydrogen bonding, hydrophobic contacts, and a salt bridge. The net positive charge on the inner surface of PCNA ring is due to the presence of several lysine and arginine residues that allows negatively charged DNA to pass through without electrostatic repulsion. The ring also has distinct front and back surface with front surface including IDCL linking N- and C- terminal domains of each monomer and back surface consisting of several loops connecting the antiparallel β-strands (Fig. 2) (Krishna et al. 1994). PCNA interacts with various binding partners on the front surface which point in the direction of DNA synthesis. These interacting partners mostly bind through a sequence, PCNA-interacting peptide (PIP) box (Seq: Q x x L/V/I/M x x F/Y/F/Y), which inserts itself into the hydrophobic pocket on the front face beneath the IDCL. Degenerated PIP box, specialized PIP box, PIP degron, reverse/inverted PIP box, and AlkB homolog 2 PCNA-interacting motif (APIM) also support PCNA interaction (Gilljam et al. 2009; Pedley et al. 2014). PCNA is an evolutionarily well-conserved protein found in all eukaryotic species from yeast to humans, as well as in archaea (Stoimenov and Helleday 2009).
Proliferating Cell Nuclear Antigen, Fig. 2

Structure of PCNA showing entire homotrimeric structure with a monomeric structure (enclosed in dotted circle)

PCNA in Physiology

PCNA plays a direct role in the metabolism of nucleic acid. However, PCNA has also been said to have a role in cytoplasmic and extracellular compartments. Most common functions of PCNA are as follows (Fig. 3).
Proliferating Cell Nuclear Antigen, Fig. 3

Interaction of PCNA with various proteins/enzymes to perform cellular activities such as DNA replication, DNA damage repair, DNA repair, chromatin assembly, sister chromatid cohesion, and cell cycle regulation

DNA Replication

In G1 phase of cell cycle, DNA replication is initiated at replication origins, marked by binding of prereplicative protein complex. The origin firing takes place in S-phase and involves assembly of two replication forks proceeding in opposite direction, each headed by enzymes that unwind the DNA. This unwinding allows the synthesis of primers by DNA polymerase (pol) α primase. Once the priming of DNA is complete, DNA polymerase δ and ε are required to complete DNA synthesis in order to finish replication. In vitro studies have shown requirement of polymerase δ on the lagging strand and polymerases ε on the leading strand for completion of replication. This exchange of polymerases at the replication fork involves PCNA assembly by temporarily breaking interactions between subunits and reassembling the ring around DNA with the help of replication factor C (RFC) protein complex. Interactions between subunits hRFC140, hRFC 36, hRFC40, and front side of PCNA in an ATP-dependent manner aid in the binding of RFC–PCNA complex to template–primer junction, thus displacing the polymerase α primase. Once the binding is completed, the RFC–PCNA complex disruption takes place due to change in RFC by ATP hydrolysis which in turn favors binding of PCNA with DNA pol δ and ε. As DNA synthesis occurs in the 5′–3′direction, replication on the lagging strand is discontinuous. Primers are being synthesized every 100–200 nucleotides to generate Okazaki fragments. Maturation of Okazaki fragments also requires assistance of PCNA. It helps in the stimulation of enzymatic activities required for the completion of replication when polymerization of newly primed fragment reaches the primer of the previous fragment. The polymerization continues through a process called nick translation and is carried out with the help of replicative polymerase in conjunction with helicase/nuclease y DNA2. The end of the previous Okazaki fragment functions as a molecular break that slows down Pol δ. The polymerase partly displaces the primer end, forming a flap structure that is cleaved off by PCNA-interacting protein flap structure-specific endonuclease (FEN) 1, one nucleotide at a time resulting in a nick which is then sealed by PCNA-interacting enzyme DNA ligase I. PCNA loading on the primer template junction at each Okazaki fragment leads to accumulation on the lagging strand. Unloading of this PCNA requires the activity of an RFC-like complex, in which the catalytic subunit hRFC140 is replaced by ATAD5 (Moldovan et al. 2007; Stoimenov and Helleday 2009; Choe and Moldovan 2017).

DNA Damage Repair

During replication if DNA damage is encountered, the replication fork may stall it by evoking various pathways involved in DNA damage avoidance. These pathways are activated through posttranslational modified PCNA molecule. Monoubiquitylation of PCNA is one such pathway that occurs at stalled replication fork and initiates signal for translesion synthesis (TLS polymerase) to act on short sequence around the damage further enabling DNA to complete replication. However, there is not much known whether there is a back switch to replicative polymerase or its TLS polymerase that fills the gap and/or behind the fork. The second pathway is not well known and involves polyubiquitylation of PCNA to cause recombination events around the fork independent of Rad 52 (Moldovan et al. 2007; Stoimenov and Helleday 2009).

DNA Repair

Stable inheritance of undamaged genetic information required the development of different types of DNA repair systems such as base excision repair (BER), nucleotide excision repair (NER), mismatch repair (MMR), and double-strand break repair (DSBR). PCNA is said to interact with repair proteins and contribute in DNA repair system. BER is responsible for replacing chemically (oxidizing, reducing, and alkylating agents) altered nucleotide bases in DNA. PCNA interacts with all proteins participating in BER and functions as bridges between proteins by stimulating and thus coordinating the process. NER usually deals with DNA lesions generated from chemical and/or ultraviolet radiation damage. In NER, PCNA bind to various proteins and facilitates new DNA fragment resynthesis. MMR helps in the correction of small insertion or deletion of loops and base–base mismatch occurring during faulty DNA replication to start a fresh synthesis. PCNA acts in the early stage of damage recognition as well as DNA incision process in MMR pathway (Moldovan et al. 2007; Stoimenov and Helleday 2009; Choe and Moldovan 2017).

Chromatin Assembly

During DNA replication, there is disruption in the assembly of chromatin structure near the replication fork. The assembly is restored once the replisome has passed leaving behind a few hundreds of nucleotide. The assembly and disassembly of chromatin is conducted by pathways that involve histone chaperone complex, chromatin assembly factor (CAF)-1, or HIR (hir1-3, Hpc2) complex and their common factor Asf1. PCNA’s interaction with CAF-1 via PIP box helps in delivering histone (H3, H4) to the replication site as well as in re-establishing chromatin after repair process is finished at NER sites. Asf1 binds to histone, thus helping CAF-1 to deliver them at newly replicated sites and HIR complex for chromosomal replication-independent chromatin assembly (Moldovan et al. 2007; Choe and Moldovan 2017).

Sister Chromatid Cohesion

For correct segregation of homologous chromosomes into two daughter cells, it is necessary that the sister chromatids are kept together throughout the G2 phase of the cell cycle until anaphase. This is mediated by a proteinaceous ring, cohesin, which is composed of the four subunits Smc1, Smc3, Scc1, and Scc3. Sister chromatid cohesion is well established in S-phase immediately after its formation by replication with the help of a protein called Eco family protein. ECO family of protein contains establishment of cohesion proteins (ESCO) 1 and 2 in humans that bind to PCNA through PIP box, thus helping build sister chromatid and progression of replication forks (Moldovan et al. 2007; Stoimenov and Helleday 2009; Choe and Moldovan 2017).

Cell Cycle Regulation

PCNA interacts with several proteins participating in cell cycle regulation. Studies have demonstrated PCNA to interact with cell division cycle kinases (CDK) 2-cyclin complex (CDK2-cyclin A, CDK2-cyclin E, etc.) during various phases of cell cycle by targeting the complex to bind to PCNA-binding DNA replication proteins and thus helping in DNA replication. PCNA also helps in the regulation of damage-induced apoptosis by interacting with PIP box of p33 (inhibitor of growth (ING) 1b), thus stimulating and inducing programmed cell death, or by interacting with growth arrest and DNA damage-inducible (Gadd) 45, myeloid differentiation factor (MyD)118, and cytokine-responsive (CR)6 proteins and suppressing their function of growth control, apoptosis, and DNA repair. PCNA has also been found to interact with p53 and its negative regulator murine double minute (Mdm) 2 and contribute indirectly in the stability of p53. PCNA is regulated by p21, a tumor-suppressor protein and potent inhibitor of CDKs, by binding through a PIP box located on it. Studies have shown that PIP box of p21 blocks the surface of PCNA required for polymerase stimulation and in process inhibiting replication. Various proteins such as Pol δ subunit, FEN-1, the licensing cofactor Cdt1, chromatin remodeling factor (WSTF), DNA methyl transferase (DNMT)1, and repair proteins are inhibited by p21 from interacting with PCNA. It also has shown to inhibit the ATPase activity of RFC along with MMR (Moldovan et al. 2007; Choe and Moldovan 2017).

PCNA in Disease

Various diseases including cancer are caused by multiple mechanisms that often arise as an error in DNA replication. Owing to its interaction with various proteins and its function per se, PCNA has been found to be associated with, and cause, various diseases/syndromes.

PCNA Mutation-Associated Syndrome

Ataxia-telangiectasia-like disorder-2 is caused by homozygous missense PCNA mutation, Ser228lle, in patients from a single extended family resulting in large conformational change that affects interactions with PIP box of proteins, FEN-1, DNA ligase 1, and XPG which in turn affects DNA repair function of PCNA. The patients with this syndrome showed symptoms varying from delayed development or learning difficulties, prelingual sensorineural hearing loss, progressive gait instability and ataxia, progressive muscle weakness, dysarthria, dysphagia, and cognitive decline with age cutaneous and conjunctival telangiectasia, photophobia, and photosensitivity. One patient had predisposition to sun-induced malignancy. However, none of the patients suffered from immunodeficiency (Baple et al. 2014).

In Diseases

PCNA was first identified as a nuclear antigen in proliferating cells, recognized by an autoantibody present in the serum of patients with systemic lupus erythematosus (Miyachi et al. 1978). In a comparative study, the percentage of PCNA expression was found to be higher in malignant skin lesions (squamous cell carcinoma, adult T lymphotrophic leukemia, mycosis fungoides, malignant melanoma, and malignant lymphoma) as compared to nonmalignant skin diseases (resistant atopic dermatitis, psoriasis vulgaris, verruca vulgaris), thus proving it to be helpful in early detection of skin malignancies (Kawahira 1999). Cancer is a multifactorial disease that may often appear due to error in DNA replication and its progression cannot be separated from proliferation and metastasis of cells. PCNA plays varied roles in DNA replication, repair, and chromatin assembly which helps in the regulation of tumor proliferation and thus also in metastasis. The cancer-specific isoform of PCNA is expressed more frequently in tumor tissue rather than its normal counterpart. However, the mechanism by which it helps in cellular malignant transformation and progression still needs further research. Several of PCNA’s binding partners have said to play a role in cancer predisposition syndrome such as ATAD5, SPARTAN, and PARI for ovarian, hepatocellular, and tumors due to genotoxic cancer therapy, respectively. In context to the role PCNA plays in cancer, it has recently become a target for cancer therapy with PCNA inhibitors being developed as potential anticancer agents. It has also been observed that ubiquitilation of PCNA causes somatic hypermutation at immunoglobulin G loci in cases of human B cell deficiency, while homozygous missense mutations in the PCNA-binding partners TRAIP and PARP10 are seen in cases of microcephaly (Choe and Moldovan 2017).

Summary

It is a puzzle how PCNA interacts and regulates so many proteins to perform its functions. Very little is known about the compartmentalization of individual interaction making it tempting to discover the role of PCNA in every individual interaction, thus helping in addressing a critical issue of its therapeutic use in the treatment of cancer. There is also the need of further research on the influences of epigenetic and genetic differences among tumors on PCNA modification.

References

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Copyright information

© Springer International Publishing AG 2018

Authors and Affiliations

  • Rashmi Maruti Hosalkar
    • 1
    • 2
  • Niharika Swain
    • 3
  • Jayesh S. Khivasara
    • 4
  • Samapika Routray
    • 5
  1. 1.Indian Association of Oral and Maxillofacial PathologistsMumbaiIndia
  2. 2.Maharashtra State Dental CouncilMumbaiIndia
  3. 3.MGM Dental College and HospitalNavi MumbaiIndia
  4. 4.Mahatma Gandhi Cancer HospitalSangliIndia
  5. 5.Department of Dental Surgery, All India Institute of Medical SciencesBhubaneswarIndia