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55-422: 5111 18538 ENSG00000132646 ENSMUSG00000027342 P12004 P17918 NM_182649 NM_002592 NM_011045 NP_002583 NP_872590 NP_035175 Proliferating cell nuclear antigen ( PCNA ) is a DNA clamp that acts as a processivity factor for DNA polymerase δ in eukaryotic cells and is essential for replication. PCNA is a homotrimer and achieves its processivity by encircling

110-465: A "Sledge Hammer" effect, in which the drug promotes cellular autophagy, part of which includes broad-spectrum promotion of nonspecific SUMOylation for many proteins. This may be beneficial in some circumstances as it supports the breakdown of accumulated waste products. The importance of these studies in models such as yeast lies in their potential to inform scientists in the research and development of precise biomedical interventions that can translate to

165-523: A C-terminal peptide is cleaved from the SUMO precursor by a protease (in human these are the SENP proteases or Ulp1 in yeast) to reveal a di-glycine motif. The obtained SUMO then becomes bound to an E1 enzyme (SUMO Activating Enzyme (SAE)) which is a heterodimer (subunits SAE1 and SAE2 ). It is then passed to an E2, which is a conjugating enzyme (Ubc9). Finally, one of a small number of E3 ligating proteins attaches it to

220-588: A DNA helicase called Srs2, which has a role in disrupting Rad51 nucleoprotein filaments fundamental for initiation of homologous recombination. PCNA interacts with many proteins. PCNA has been shown to interact with: Proteins interacting with PCNA via APIM include human AlkB homologue 2, TFIIS-L, TFII-I, Rad51B, XPA, ZRANB3, and FBH1. Antibodies against proliferating cell nuclear antigen (PCNA) or monoclonal antibody termed Ki-67 can be used for grading of different neoplasms , e.g. astrocytoma . They can be of diagnostic and prognostic value. Imaging of

275-468: A clamp loader and with a polymerase. Thus the clamp will not be actively disassembled while the polymerase remains bound. DNA clamps also associate with other factors involved in DNA and genome homeostasis, such as nucleosome assembly factors, Okazaki fragment ligases, and DNA repair proteins. All of these proteins also share a binding site on the DNA clamp that overlaps with the clamp loader site, ensuring that

330-516: A closed ring encircling duplex DNA. Certain NSAIDs (carprofen, bromfenac, and vedaprofen) exhibit some suppression of bacterial DNA replication by inhibiting bacterial DNA clamp. The sliding clamp in eukaryotes is assembled from a specific subunit of DNA polymerase delta called the proliferating cell nuclear antigen ( PCNA ). The N-terminal and C-terminal domains of PCNA are topologically identical. Three PCNA molecules are tightly associated to form

385-459: A closed ring encircling duplex DNA. The sequence of PCNA is well conserved between plants, animals and fungi, indicating a strong selective pressure for structure conservation, and suggesting that this type of DNA replication mechanism is conserved throughout eukaryotes. In eukaryotes, a homologous, heterotrimeric "9-1-1 clamp" made up of RAD9 - RAD1 - HUS1 (911) is responsible for DNA damage checkpoint control. This 9-1-1 clamp mounts onto DNA in

440-459: A critical component of the DNA polymerase III holoenzyme , the clamp protein binds DNA polymerase and prevents this enzyme from dissociating from the template DNA strand. The clamp-polymerase protein–protein interactions are stronger and more specific than the direct interactions between the polymerase and the template DNA strand; because one of the rate-limiting steps in the DNA synthesis reaction

495-439: A high degree of similarity to each other and are distinct from SUMO-1. SUMO-4 shows similarity to SUMO-2/3 but differs in having a Proline instead of Glutamine at position 90. As a result, SUMO-4 isn't processed and conjugated under normal conditions, but is used for modification of proteins under stress-conditions like starvation. During mitosis, SUMO-2/3 localize to centromeres and condensed chromosomes, whereas SUMO-1 localizes to

550-610: A molecular glue to facilitate the assembly of large protein complexes in repair foci. Also, SUMOylation can alter a protein's biochemical activities and interactions. SUMOylation plays a role in the major DNA repair pathways of base excision repair , nucleotide excision repair , non-homologous end joining and homologous recombinational repair. SUMOylation also facilitates error prone translation synthesis. SUMO proteins are small; most are around 100 amino acids in length and 12 kDa in mass . The exact length and mass varies between SUMO family members and depends on which organism

605-412: A multimeric, six-domain ring structure that completely encircles the DNA double helix as the polymerase adds nucleotides to the growing strand. Each domain is in turn made of two β-α-β-β-β structural repeats. The DNA clamp assembles on the DNA at the replication fork and "slides" along the DNA with the advancing polymerase, aided by a layer of water molecules in the central pore of the clamp between

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660-413: A ring, it does not need a clamp loader to be attached to DNA. Sliding clamps are loaded onto their associated DNA template strands by specialized proteins known as " sliding clamp loaders ", which also disassemble the clamps after replication has completed. The binding sites for these initiator proteins overlap with the binding sites for the DNA polymerase, so the clamp cannot simultaneously associate with

715-471: A small fraction of a given protein is SUMOylated and this modification is rapidly reversed by the action of deSUMOylating enzymes. SUMOylation of target proteins has been shown to cause a number of different outcomes including altered localization and binding partners. The SUMO-1 modification of RanGAP1 (the first identified SUMO substrate) leads to its trafficking from cytosol to nuclear pore complex. The SUMO modification of ninein leads to its movement from

770-534: A substantial component of fundamental biology. SUMO proteins are similar to ubiquitin and are considered members of the ubiquitin-like protein family. SUMOylation is directed by an enzymatic cascade analogous to that involved in ubiquitination. In contrast to ubiquitin, SUMO is not used to tag proteins for degradation . Mature SUMO is produced when the last four amino acids of the C-terminus have been cleaved off to allow formation of an isopeptide bond between

825-420: Is a potential therapeutic target in cancer therapy. In 2023 City of Hope National Medical Center published preclinical research on a targeted chemotherapy using AOH1996 that appears to suppress tumor growth without causing discernable side effects. DNA clamp A DNA clamp , also known as a sliding clamp , is a protein complex that serves as a processivity -promoting factor in DNA replication . As

880-481: Is a regulatory ubiquitin ligase protein in humans called Mouse Double Minute 2 protein, or MDM2, which acts to remove p53 from the cell. MDM2 regulates itself through self-ubiquitination by way of a RING finger domain, targeting itself for proteasomal destruction. When it is SUMOylated at the RING finger domain , MDM2 no longer limits its own function in the cell. When protected from itself, it likewise ubiquitinates p53, marking

935-417: Is called SUMOylation (pronounced soo-muh-lā-shun and sometimes written sumoylation ). SUMOylation is a post-translational modification involved in various cellular processes, such as nuclear - cytosolic transport, transcriptional regulation, apoptosis , protein stability, response to stress, and progression through the cell cycle . In human proteins, there are over 53,000 SUMO binding sites, making it

990-441: Is involved in resynthesis of excised damaged DNA strands during DNA repair , PCNA is important for both DNA synthesis and DNA repair. PCNA is also involved in the DNA damage tolerance pathway known as post-replication repair (PRR). In PRR, there are two sub-pathways: (1) a translesion synthesis pathway, which is carried out by specialised DNA polymerases that are able to incorporate damaged DNA bases into their active sites (unlike

1045-451: Is referred to as the beta clamp. In archaea and eukaryotes, it is a trimer composed of three molecules of PCNA . The T4 bacteriophage also uses a sliding clamp, called gp45 that is a trimer similar in structure to PCNA but lacks sequence homology to either PCNA or the bacterial beta clamp. The beta clamp is a specific DNA clamp and a subunit of the DNA polymerase III holoenzyme found in bacteria. Two beta subunits are assembled around

1100-446: Is the lysine conjugated to SUMO, x is any amino acid (aa), D or E is an acidic residue. Substrate specificity appears to be derived directly from Ubc9 and the respective substrate motif. Currently available prediction programs are: SUMO attachment to its target is similar to that of ubiquitin (as it is for the other ubiquitin-like proteins such as NEDD 8). The SUMO precursor has some extra amino acids that need to be removed, therefore

1155-402: Is the association of the polymerase with the DNA template, the presence of the sliding clamp dramatically increases the number of nucleotides that the polymerase can add to the growing strand per association event. The presence of the DNA clamp can increase the rate of DNA synthesis up to 1,000-fold compared with a nonprocessive polymerase. The DNA clamp is an α+β protein that assembles into

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1210-489: The cell cycle . Part of the protein was sequenced and that sequence was used to allow isolation of a cDNA clone. PCNA helps hold DNA polymerase delta ( Pol δ ) to DNA. PCNA is clamped to DNA through the action of replication factor C (RFC), which is a heteropentameric member of the AAA+ class of ATPases. Expression of PCNA is under the control of E2F transcription factor -containing complexes. Since DNA polymerase epsilon

1265-459: The centrosome to the nucleus . In many cases, SUMO modification of transcriptional regulators correlates with inhibition of transcription. One can refer to the GeneRIFs of the SUMO proteins, e.g. human SUMO-1, to find out more. There are 4 confirmed SUMO isoforms in humans; SUMO-1 , SUMO-2 , SUMO-3 and SUMO-4 . At the amino acid level, SUMO1 is about 50% identical to SUMO2. SUMO-2/3 show

1320-556: The C-terminal glycine residue of SUMO and an acceptor lysine on the target protein. SUMO family members often have dissimilar names; the SUMO homologue in yeast , for example, is called SMT3 (suppressor of mif two 3). Several pseudogenes have been reported for SUMO genes in the human genome . SUMO modification of proteins has many functions. Among the most frequent and best studied are protein stability, nuclear - cytosolic transport, and transcriptional regulation. Typically, only

1375-628: The Cohesin protein which binds sister chromatids in metaphase is able to be targeted by SUMOylation to allow the Cdc48 segregase to separate Cohesin and allow sister chromatid separation in early anaphase. In research as is often the case, scientists test drugs known to have significant effects on living systems; one such example is Rapamycin (known in pharmaceuticals as Sirolimus ), the well-known inhibitor of mechanistic Target of Rapamycin, or mTOR . With respect to SUMOylation, Rapamycin may be thought of as having

1430-399: The DNA and the protein surface. Because of the toroidal shape of the assembled multimer, the clamp cannot dissociate from the template strand without also dissociating into monomers . The DNA clamp fold is found in bacteria , archaea , eukaryotes and some viruses . In bacteria, the sliding clamp is a homodimer composed of two identical beta subunits of DNA polymerase III and hence

1485-546: The DNA by the gamma subunit and ATP hydrolysis; this assembly is called the pre-initiation complex . After assembly around the DNA, the beta subunits' affinity for the gamma subunit is replaced by an affinity for the alpha and epsilon subunits, which together create the complete holoenzyme. DNA polymerase III is the primary enzyme complex involved in prokaryotic DNA replication . The gamma complex of DNA polymerase III, composed of γδδ'χψ subunits, catalyzes ATP to chaperone two beta subunits to bind to DNA. Once bound to DNA,

1540-497: The DNA, where it acts as a scaffold to recruit proteins involved in DNA replication, DNA repair, chromatin remodeling and epigenetics . Many proteins interact with PCNA via the two known PCNA-interacting motifs PCNA-interacting peptide (PIP) box and AlkB homologue 2 PCNA interacting motif (APIM). Proteins binding to PCNA via the PIP-box are mainly involved in DNA replication whereas proteins binding to PCNA via APIM are mainly important in

1595-585: The E3 ligases). SUMOylation is reversible and is removed from targets by specific SUMO proteases. In budding yeast, the Ulp1 SUMO protease is found bound at the nuclear pore, whereas Ulp2 is nucleoplasmic. The distinct subnuclear localisation of deSUMOylating enzymes is conserved in higher eukaryotes. SUMO can be removed from its substrate, which is called deSUMOylation. Specific proteases mediate this procedure (SENP in human or Ulp1 and Ulp2 in yeast). In yeast, SMT3 encodes

1650-589: The PIP (PCNA-interacting protein) motif, but a wider variety of such proteins performing different functions are found. PCNA is also appropriated by some viruses. The giant virus genus Chlorovirus , with PBCV-1 as a representative, carries in its genome two PCNA genes ( Q84513 , O41056 ) and a eukaryotic-type DNA polymerase. Members of Baculoviridae also encode a PCNA homolog ( P11038 ). The viral gp45 sliding clamp subunit protein contains two domains. Each domain consists of two alpha helices and two beta sheets –

1705-478: The SUMO protein, and SUMO E3 ligase attaches SUMO to target proteins. In cell cycle regulation, the base case is that SUMO ligation is constantly taking place, leading to polySUMOylation of eligible target proteins. This is countered by the SUMO protease Ulp2 which cleaves polySUMO groups, leaving the protein in a monoSUMOylated state. As shown in the Biorender figure, there is a feedback mechanism in which ULP2 maintains

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1760-505: The Smc5/6 complex) and Pias-gamma and HECT proteins. On Chromosome 17 of the human genome, SUMO2 is near SUMO1+E1/E2 and SUMO2+E1/E2, among various others. Some E3's, such as RanBP2, however, are neither. Recent evidence has shown that PIAS-gamma is required for the SUMOylation of the transcription factor yy1 but it is independent of the zinc-RING finger (identified as the functional domain of

1815-420: The beta subunits can freely slide along double stranded DNA. The beta subunits in turn bind the αε polymerase complex. The α subunit possesses DNA polymerase activity and the ε subunit is a 3’-5’ exonuclease . The beta chain of bacterial DNA polymerase III is composed of three topologically equivalent domains ( N-terminal , central, and C-terminal ). Two beta chain molecules are tightly associated to form

1870-445: The clamp will not be removed while any enzyme is still working on the DNA. The activity of the clamp loader requires ATP hydrolysis to "close" the clamp around the DNA. Small ubiquitin-like modifier In molecular biology , SUMO ( S mall U biquitin-like Mo difier) proteins are a family of small proteins that are covalently attached to and detached from other proteins in cells to modify their function. This process

1925-474: The context of genotoxic stress. The protein encoded by this gene is found in the nucleus and is a cofactor of DNA polymerase delta. The encoded protein acts as a homotrimer and helps increase the processivity of leading strand synthesis during DNA replication. In response to DNA damage, this protein is ubiquitinated and is involved in the RAD6-dependent DNA repair pathway. Two transcript variants encoding

1980-547: The counteracting deSUMOylation is the following: First, the targeted protein becomes polySUMOylated. Second, SUMO Targeted Ubiquitin Ligase , or STUbL, (SLX5 or SLX8 in the case of yeast) may then bind the polySUMOylated target and attach Ubiquitin groups (often polyUbiquitinating the already polySUMOylated protein). Third, segregases such as Cdc48 may then dissociate the SUMOylated and ubiquitinated target from its bound protein. Fourth,

2035-508: The deSUMOylation of Ulp2, polySUMOylation is then promoted as the new stable state of target proteins, which are often but not always bound to other proteins in order to regulate major changes within the cell. Cdc5 is countered by the Rts1-PP2A phosphatase, which maintains the active state of the Ulp2 SUMO protease by removing the phosphate group added by Cdc5 kinase. The consequence of disrupting

2090-481: The dissociated protein may then be degraded by the canonical Ubiquitin- Proteasome pathway while the unbound protein it had been bound to is now free to do what it could not do while bound. As studied with budding yeast, in the case of Tof2-Cdc14, Cdc14 release from the nucleolus allows the Mitotic Exit Network to commence, but it is regulated by the binding of Tof2, a protein subject to SUMOylation. Likewise,

2145-415: The fold is duplicated and has internal pseudo two-fold symmetry. Three gp45 molecules are tightly associated to form a closed ring encircling duplex DNA. Some members of Herpesviridae encode a protein that has a DNA clamp fold but does not associate into a ring clamp. The two-domain protein does, however, associate with the viral DNA polymerase and also acts to increase processivity. As it does not form

2200-440: The improvement of human health in an array of clinical aspects. SUMO protein is implicated in the etiology of many biomedical disease states not limited to: cancer, diabetes, chronic inflammatory tumors, neurodegenerative diseases, cardiovascular diseases, pulmonary diseases, atherosclerosis, liver diseases, infectious diseases, and intestinal disorders. In the case of the well-studied cancer tumor suppressor known as p53, there

2255-515: The internal SUMO consensus sites found in SUMO-2/3, it is thought to terminate these poly-SUMO chains. Serine 2 of SUMO-1 is phosphorylated, raising the concept of a 'modified modifier'. Cellular DNA is regularly exposed to DNA damaging agents. A DNA damage response (DDR) that is well regulated and intricate is usually employed to deal with the potential deleterious effects of the damage. When DNA damage occurs, SUMO protein has been shown to act as

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2310-483: The mitotic spindle and spindle midzone, indicating that SUMO paralogs regulate distinct mitotic processes in mammalian cells. One of the major SUMO conjugation products associated with mitotic chromosomes arose from SUMO-2/3 conjugation of topoisomerase II, which is modified exclusively by SUMO-2/3 during mitosis. SUMO-2/3 modifications seem to be involved specifically in the stress response. SUMO-1 and SUMO-2/3 can form mixed chains, however, because SUMO-1 does not contain

2365-475: The monoSUMOylated state by passively and diligently cleaving SUMO such that the polySUMOyated state is never stabilized enough to be acted upon by downstream actors. This deSUMOylation is critical to prevent precocious advancement of the cell cycle as discussed in several studies. The deSUMOylation may be arrested by the inhibitory phosphorylation of the Ulp2 SUMO protease by the Polo-like kinase Cdc5. By inhibiting

2420-419: The need for transfection and bypass the problem of difficult to transfect and/or short lived cells, cell permeable replication and/or repair markers can be used. These peptides offer the distinct advantage that they can be used in situ in living tissue and even distinguish cells undergoing replication from cells undergoing repair. caPCNA, a post-translationally modified isoform of PCNA common in cancer cells,

2475-446: The normal replicative polymerase, which stall), and hence bypass the damage, and (2) a proposed "template switch" pathway that is thought to involve damage bypass by recruitment of the homologous recombination machinery. PCNA is pivotal to the activation of these pathways and the choice as to which pathway is utilised by the cell. PCNA becomes post-translationally modified by ubiquitin . Mono-ubiquitin of lysine number 164 on PCNA activates

2530-413: The nuclear distribution of PCNA (via antibody labeling) can be used to distinguish between early, mid and late S phase of the cell cycle. However, an important limitation of antibodies is that cells need to be fixed leading to potential artifacts. On the other hand, the study of the dynamics of replication and repair in living cells can be done by introducing translational fusions of PCNA. To eliminate

2585-405: The nucleus, which prevents such activity. The critical nature of p53 cannot be overstated: in fact, if a human carries only one non-functioning copy of p53, it results in a deadly cancer prognosis known as Li Fraumeni syndrome . Recombinant proteins expressed in E. coli may fail to fold properly, instead forming aggregates and precipitating as inclusion bodies . This insolubility may be due to

2640-434: The opposite direction. Archaea , probable evolutionary precursor of eukaryotes, also universally have at least one PCNA gene. This PCNA ring works with PolD , the single eukaryotic-like DNA polymerase in archaea responsible for multiple functions from replication to repair. Some unusual species have two or even three PCNA genes, forming heterotrimers or distinct specialized homotrimers. Archaeons also share with eukaryotes

2695-408: The presence of codons read inefficiently by E. coli , differences in eukaryotic and prokaryotic ribosomes, or lack of appropriate molecular chaperones for proper protein folding. In order to purify such proteins it may be necessary to fuse the protein of interest with a solubility tag such as SUMO or MBP ( maltose-binding protein ) to increase the protein's solubility. SUMO can later be cleaved from

2750-432: The protective p53 for destruction instead, whose absence is understood to promote cancer. Here again, the base case is SUMOylation, which is actively being undone by newly discovered SUMO protease SUSP4 and also by the SUMO protease interaction of SMT3IP1/SENP3 which is understood to deSUMOylate both MDM2 and p53. One of the ways p53 functions is as a DNA-binding tetramer; interestingly, SUMOylation of p53 delocalizes it from

2805-400: The protein comes from. Although SUMO has very little sequence identity with ubiquitin (less than 20%) at the amino acid level, it has a nearly identical structural fold. SUMO protein has a unique N-terminal extension of 10-25 amino acids which other ubiquitin-like proteins do not have. This N-terminal is found related to the formation of SUMO chains. The structure of human SUMO1 is depicted on

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2860-563: The protein. In budding yeast, there are four SUMO E3 proteins, Cst9, Mms21, Siz1 and Siz2 . While in ubiquitination an E3 is essential to add ubiquitin to its target, evidence suggests that the E2 is sufficient in SUMOylation as long as the consensus sequence is present. It is thought that the E3 ligase promotes the efficiency of SUMOylation and in some cases has been shown to direct SUMO conjugation onto non-consensus motifs. E3 enzymes can be largely classed into PIAS proteins, such as Mms21 (a member of

2915-415: The right. It shows SUMO1 as a globular protein with both ends of the amino acid chain (shown in red and blue) sticking out of the protein's centre. The spherical core consists of an alpha helix and a beta sheet . The diagrams shown are based on an NMR analysis of the protein in solution. Most SUMO-modified proteins contain the tetrapeptide consensus motif Ψ-K-x-D/E where Ψ is a hydrophobic residue, K

2970-399: The same protein have been found for this gene. Pseudogenes of this gene have been described on chromosome 4 and on the X chromosome. PCNA is also found in archaea, as a processivity factor of polD , the single multi-functional DNA polymerase in this domain of life. PCNA was originally identified as an antigen that is expressed in the nuclei of cells during the DNA synthesis phase of

3025-413: The translesion synthesis pathway. Extension of this mono-ubiquitin by a non-canonical lysine-63-linked poly-ubiquitin chain on PCNA is thought to activate the template switch pathway. Furthermore, sumoylation (by small ubiquitin-like modifier , SUMO) of PCNA lysine-164 (and to a lesser extent, lysine-127) inhibits the template switch pathway. This antagonistic effect occurs because sumoylated PCNA recruits

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