Proliferating cell nuclear antigen


PCNA and DNA repair

Nucleotide excision repair is the principal way by which human cells remove UV damage from DNA. Human cell extracts were fractionated to locate active components, including xeroderma pigmentosum (XP) and ERCC factors. The incision reaction was then reconstituted with the purified proteins RPA, XPA, TFIIH (containing XPB and XPD), XPC, UV-DDB, XPG, partially purified ERCC1/XPF complex, and a factor designated IF7. UV-DDB (related to XPE protein) stimulated repair but was not essential. ERCC1- and XPF-correcting activity copurifies with an ERCC1-binding polypeptide of 110 kDa that is absent in XP-F cell extract. Complete repair synthesis is achieved by combining these factors with DNA polymerase epsilon, RFC, PCNA, and DNA ligase I. The reconstituted core reaction requires about 30 polypeptides (Aboussekhra, 1995).

In eukaryotic cells, a 5' flap DNA endonuclease activity and a ds DNA 5'-exonuclease activity exist within a single enzyme called FEN-1 [flap endo-nuclease and 5(five)'-exo-nuclease]. This 42 kDa endo-/exonuclease, FEN-1, is highly homologous to human XP-G, Saccharomyces cerevisiae RAD2 and S.cerevisiae RTH1. These structure-specific nucleases recognize and cleave a branched DNA structure called a DNA flap, and its derivative, called a pseudo Y-structure. FEN-1 is essential for lagging strand DNA synthesis in Okazaki fragment joining. FEN-1 also appears to be important in mismatch repair. Human PCNA, the processivity factor for eukaryotic polymerases, physically associates with human FEN-1 and stimulates its endonucleolytic activity at branched DNA structures and its exonucleolytic activity at nick and gap structures. Structural requirements for FEN-1 and PCNA loading provide an interesting picture of this stimulation. PCNA loads on to substrates at double-stranded DNA ends. In contrast, FEN-1 requires a free single-stranded 5' terminus and appears to load by tracking along the single-stranded DNA branch. These physical constraints define the range of DNA replication, recombination and repair processes in which this family of structure-specific nucleases participate. A model explaining the exonucleolytic activity of FEN-1 in terms of its endonucleolytic activity is proposed based on these observations (Wu, 1996).

Two forms of DNA base excision-repair (BER) have been observed: a 'short-patch' BER pathway involving replacement of one nucleotide and a 'long-patch' BER pathway with gap-filling of several nucleotides. The latter mode of repair has been investigated using human cell-free extracts or purified proteins. Correction of a regular abasic site in DNA mainly involves incorporation of a single nucleotide, whereas repair patches of two to six nucleotides in length are found after repair of a reduced or oxidized abasic site. Human AP endonuclease, DNA polymerase beta and a DNA ligase (either III or I) are sufficient for the repair of a regular AP site. In contrast, the structure-specific nuclease DNase IV (FEN1) is essential for repair of a reduced AP site, which occurs through the long-patch BER pathway. DNase IV is required for cleavage of an reaction intermediate, generated by template strand displacement during gap-filling. XPG, a related nuclease, can not substitute for DNase IV. The long-patch BER pathway is largely dependent on DNA polymerase beta in cell extracts, but the reaction can be reconstituted with either DNA polymerase beta or delta. Efficient repair of gamma-ray-induced oxidized AP sites in plasmid DNA also requires DNase IV. PCNA can promote the Pol beta-dependent long-patch pathway by stimulation of DNase IV (Klungland, 1997).

In vitro studies have suggested a role for PCNA-in the repair synthesis step of nucleotide excision repair. PCNA interacts with the cyclin-dependent kinase inhibitor p21, but due to the lack of genetic evidence, it is not clear which of the DNA repair processes are in fact affected by PCNA in vivo. A PCNA mutation (pol30-46) confers ultraviolet (UV) sensitivity but has no effect on growth or cell cycle progression; the mutant pcna interacts normally with DNA polymerase delta and epsilon. Genetic studies indicate that the pol30-46 mutation is specifically defective in RAD6-dependent postreplicational repair of UV damaged DNA; this mutation impairs the error-free mode of bypass repair. These results implicate a role for PCNA as an intermediary between DNA replication and postreplicational DNA repair (Torres-Ramos, 1996).

DNA lesions in the template strand pose a block to the replication machinery. Replication across such lesions may occur by a mutagenic bypass process in which a wrong base is inserted opposite the lesion or may involve processes that are relatively error-free. Genetic studies in the yeast Saccharomyces cerevisiae have indicated the requirement of REV3-encoded DNA polymerase in mutagenic bypass. The DNA polymerase responsible for error-free bypass, however, has not been identified, but genetic studies implicating proliferating cell nuclear antigen in this process have suggested that either DNA polymerase delta or DNA polymerase epsilon may be involved. This study investigated temperature-sensitive (ts) conditional lethal mutations of S. cerevisiae POL2 and POL3 genes, which encode DNA polymerase epsilon and delta, respectively. Post-replicational bypass of UV-damaged DNA is severely inhibited in the pol3-3 mutant at the restrictive temperature. By contrast, the pol-2-18 mutation has no adverse effect on this process at the restrictive temperature. From these observations, it is inferred that there is a requirement of DNA polymerase delta in post-replicative bypass of UV-damaged DNA (Torres-Ramos, 1997).

p21, a p53-induced gene product that blocks cell cycle progression at the G1 phase, interacts with both cyclin-dependent kinases and proliferating cell nuclear antigen (PCNA). PCNA functions as a processivity factor for DNA polymerases delta and epsilon and is required for both DNA replication and nucleotide excision repair. p21 inhibits simian virus 40 (SV40) DNA replication in HeLa cell extracts by interacting with PCNA. p21 blocks nucleotide excision repair of DNA that has been damaged by either ultraviolet radiation or alkylating agents; this inhibition can be reversed following addition of PCNA. p21 is more effective in blocking DNA resynthesis than in inhibiting the excision step. A peptide derived from the carboxyl terminus of p21, which specifically interacts with PCNA, inhibits polymerase delta-catalyzed elongation of DNA chains almost stoichiometrically relative to the concentration of PCNA. When added at higher levels, this peptide also blocks both SV40 DNA replication and nucleotide excision repair in HeLa cell extracts. These results indicate that p21 interferes with the function of PCNA in both in vitro DNA replication and nucleotide excision repair (Pan, 1995).

DNA damage frequently leads to the production of apurinic/apyrimidinic (AP) sites, which are presumed to be repaired through the base excision pathway. For detailed analyses of this repair mechanism, a synthetic analog of an AP site, 3-hydroxy-2-hydroxymethyltetrahydrofuran (tetrahydrofuran), was employed in a model system. Tetrahydrofuran residues were efficiently repaired in a Xenopus laevis oocyte extract in which most repair events involved ATP-dependent incorporation of no more than four nucleotides. Using a series of column chromatography procedures to fractionate X. laevis ovarian extracts, a reconstituted system was developed of tetrahydrofuran repair with five fractions, three of which were purified to near homogeneity: proliferating cell nuclear antigen (PCNA), AP endonuclease, and DNA polymerase delta. This PCNA-dependent system repairs natural AP sites as well as tetrahydrofuran residues. DNA polymerase beta is able to replace DNA polymerase delta only for repair of natural AP sites in a reaction that does not require PCNA. DNA polymerase alpha does not support repair of either type of AP site. This result indicates that AP sites can be repaired by two distinct pathways: the PCNA-dependent pathway and the DNA polymerase beta-dependent pathway (Matsumoto, 1994).

To identify in vivo pathways that compensate for impaired proliferating cell nuclear antigen (PCNA or Pol30p in yeast) activity, a synthetic lethal screen was performed with the yeast pol30-104 mutation. Nine mutations were identified that display synthetic lethality with pol30-104; three mutations affect the structural gene for the large subunit of replication factor C (rfc1), which loads PCNA onto DNA, and six mutations affect three members of the RAD52 epistasis group for DNA recombinational repair (rad50, rad52 and rad57). pol30-104 is also found to displayed synthetic lethality with mutations in other members of the RAD52 epistasis group (rad51 and rad54 - Drosophila homolog: Okra), but not with mutations in members of the RAD3 nor the RAD6 epistasis group. Analysis of nine different pol30 mutations shows that the requirement for the RAD52 pathway is correlated with a DNA replication defect but not with the relative DNA repair defect caused by pol30 mutations. Mutants that require RAD52 for viability (pol30-100, pol30-104, rfc1-1 and rth1delta) accumulate small single-stranded DNA fragments during DNA replication in vivo. Taken together, these data suggest that the RAD52 pathway is required when there are defects in the maturation of Okazaki fragments (Merrill, 1998).

DNA ligase I belongs to a family of proteins that bind to proliferating cell nuclear antigen (PCNA) via a conserved 8-amino-acid motif. The biological significance of this interaction has been examined. Inactivation of the PCNA-binding site of DNA ligase I has no effect on its catalytic activity or its interaction with DNA polymerase ß. In contrast, the loss of PCNA binding severely compromises the ability of DNA ligase I to join Okazaki fragments. Thus, the interaction between PCNA and DNA ligase I is not only critical for the subnuclear targeting of the ligase, but also for coordination of the molecular transactions that occur during lagging-strand synthesis. A functional PCNA-binding site is also required for the ligase to complement hypersensitivity of the DNA ligase I mutant cell line 46BR.1G1 to monofunctional alkylating agents, indicating that a cytotoxic lesion is repaired by a PCNA-dependent DNA repair pathway. Extracts from 46BR.1G1 cells are defective in long-patch, but not short-patch, base-excision repair (BER: short-patch BER is defined as one-nucleotide repair and long-patch BER as repair tract of 2-11 nucleotides). These results show that the interaction between PCNA and DNA ligase I has a key role in long-patch BER and provide the first evidence for the biological significance of this repair mechanism (Levin, 2000).

To gain insight into the function and organization of proteins assembled on the DNA in response to genotoxic insult, the phosphorylation of the Schizosaccharomyces pombe PCNA-like checkpoint protein Rad9 was investigated. C-terminal T412/S423 phosphorylation of Rad9 by Rad3ATR occurs in S phase without replication stress. Rad3ATR and Tel1ATM phosphorylate these same residues, plus additional ones, in response to DNA damage. In S phase and after damage, only Rad9 phosphorylated on T412/S423, but not unphosphorylated Rad9, associates with a two-BRCT-domain region of the essential Rad4TOPBP1 protein. Rad9-Rad4TOPBP1 interaction is required to activate the Chk1 damage checkpoint but not the Cds1 replication checkpoint. When the Rad9-T412/S423 are phosphorylated, Rad4TOPBP1 coprecipitates with Rad3ATR, suggesting that phosphorylation coordinates formation of an active checkpoint complex (Furuya, 2004).

Most of the proteins involved in the DNA damage and DNA replication checkpoint have been identified, and the majority are highly conserved through evolution. Many features of the checkpoint pathways remain unexplained, including their apparent complexity and the fact that many of the same proteins participate in both the DNA damage and DNA replication responses. The data presented in this study begin to uncover the molecular organization of the checkpoint proteins following their recruitment to sites of DNA damage or collapsed DNA replication forks. It is suggested that one of the reasons for the apparent complexity of the system is because it allows cells to distinguish between similar biochemical consequences of DNA damage (such as ssDNA-RPA complexes) that occurs in distinct circumstances (such as induced damage in G2 and collapsed replication forks). It is important to make these distinctions because different signaling responses to the cell cycle and the DNA repair apparatus will be appropriate in each case. The data suggest that the Rad3ATR-dependent phosphorylation of Rad9 promotes association between Rad9 and Rad4TOPBP1 through phospho-specific BRCT-domain interactions during unperturbed S phase, and that this helps cells distinguish collapsed forks from DNA damage in G2 cells. It is intriguing that a phospho-specific BRCT-mediated interaction between BRCA1 and BACH1 in human cells is promoted by cyclin-dependent kinase activity against BACH1 in G2 and also by the G2 checkpoint. Together, these observations suggest that a combination of phosphorylation events can orchestrate the organization of the checkpoint apparatus before it is activated and that, upon activation, the consequent phospho-specific protein interactions dictate the downstream consequences of this activation (Furuya, 2004).

Chk1 is a kinase crucial for genomic integrity and an effector of ATR (ATM and Rad3-realated) in DNA damage response. This study shows that Chk1 regulates the DNA damage-induced ubiquitination of proliferating cell nuclear antigen (PCNA), which facilitates the continuous replication of damaged DNA. Surprisingly, this Chk1 function requires the DNA replication protein Claspin but not ATR. Claspin, which is stabilized by Chk1, regulates the binding of the ubiquitin ligase Rad18 to chromatin. Timeless, a Claspin-associating protein, is also required for efficient PCNA ubiquitination. Thus, Chk1 and the Claspin-Timeless module of replication forks not only participate in ATR signaling, but also protect stressed forks independently of ATR (Yang, 2008).

PCNA and differentiation

The distribution of PCNA in specific somatic and germ cells of the adult mouse ovary and testis was assessed using immunocytochemical staining and immunoblot analysis and was correlated with cellular proliferation and differentiation. In the adult ovary, immunocytochemical staining for PCNA within follicular cells varies depending on the stage of follicular growth. Since PCNA staining has proven to be a useful indicator of cells involved in DNA synthesis and repair, the pattern of PCNA staining in the ovary was compared to previous studies that used tritiated thymidine labeling as a marker for DNA synthesis. In the testis, PCNA is detected in the mitotically proliferating spermatogonia, but not in spermatocytes which have just entered meiosis. PCNA staining is again observed in spermatogenic cells in later stages of meiotic prophase, in particular zygotene and pachytene spermatocytes. Since these cells are undergoing meiotic recombination, the presence of PCNA in these meiotic prophase cells could reflect a second function of PCNA, that of DNA excision repair (Chapman, 1994).

Protein levels of cyclin D3 are highly detectable in rat thymus and testis. Since testis offer unique opportunities to examine the cell cycle in vivo, the temporal and spatial expression of cyclin D3 and proliferating cell nuclear antigen (known as PCNA, the DNA synthesis indicator) were studied in the rat testis during development. The protein levels of cyclin D3 protein in testis from 7 days to 3 months old are almost constant, decreasing gradually thereafter. The protein levels of cyclin D1 and PCNA are high in the testis of 7- and 14-day-old rats and decrease during testicular development. In the seminiferous tubules of 7-day-old newborns, cyclin D3 is surprisingly located in the cytoplasm of stem cells that have bigger nuclei than the nuclei of surrounding cells. Interestingly, cyclin D3 immunopositive cells do not immunostain with PCNA in nuclei. In the adult testis, anti-cyclin D3 antibody strongly stains the cytoplasm of early stage primary spermatocytes, lightly stains pachytene spermatocytes, but does not stain elongated spermatids. There is no detectable cyclin D3 in Sertoli cells, interstitial cells, or fibroblasts within seminiferous tubules, or in blood vessels within the interstitial matrix. The known cyclin D3 partner, cyclin dependent kinase 4, is located mainly in nuclei of spermatogonia and in early stage primary spermatocytes. Strong PCNA immunopositive staining is located in the nuclei of spermatogonia in adult testis. These results indicate that cyclin D3 is detectable in meiotically active male germ cells (PCNA-negative cells), but is conspicuously absent from mitotically active spermatogonia (PCNA-positive cells). In contrast to in vitro reports, cyclin D3 is not located in the nucleus, but rather in the cytoplasm of male germ cells in vivo. Taken together, the presence of cyclin D3 in spermatocytes and its location in the cytoplasm leads to the speculation that cyclin D3 may have functions in male germ cells other than mitosis (Kang, 1997).

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Proliferating cell nuclear antigen: Biological Overview | Regulation | Developmental Biology | Effects of Mutation | References

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