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

Gene name - Proliferating cell nuclear antigen

Synonyms - mutagen-sensitive 209

Cytological map position - 56F5--56F5

Function - Auxiliary protein for DNA polymerase

Keywords - DNA replication

Symbol - PCNA

FlyBase ID: FBgn0005655

Genetic map position - 2-92.3

Classification - proliferating cell nuclear antigen

Cellular location - nuclear



NCBI links: Precomputed BLAST | Entrez Gene
BIOLOGICAL OVERVIEW

Recent literature
Huang, F., Saraf, A., Florens, L., Kusch, T., Swanson, S. K., Szerszen, L. T., Li, G., Dutta, A., Washburn, M. P., Abmayr, S. M. and Workman, J. L. (2016). The Enok acetyltransferase complex interacts with Elg1 and negatively regulates PCNA unloading to promote the G1/S transition. Genes Dev 30: 1198-1210. PubMed ID: 27198229
Summary:
KAT6 histone acetyltransferases (HATs) are highly conserved in eukaryotes and are involved in cell cycle regulation. However, information regarding their roles in regulating cell cycle progression is limited. This study reports the identification of subunits of the Drosophila Enok complex and demonstrates that all subunits are important for its HAT activity. A novel interaction is reported between the Enok complex and the Elg1 proliferating cell nuclear antigen (PCNA)-unloader complex. Depletion of Enok in S2 cells resulted in a G1/S cell cycle block, and this block can be partially relieved by depleting Elg1. Furthermore, depletion of Enok reduced the chromatin-bound levels of PCNA in both S2 cells and early embryos, suggesting that the Enok complex may interact with the Elg1 complex and down-regulate its PCNA-unloading function to promote the G1/S transition. Supporting this hypothesis, depletion of Enok also partially rescued the endoreplication defects in Elg1-depleted nurse cells. Taken together, this study provides novel insights into the roles of KAT6 HATs in cell cycle regulation through modulating PCNA levels on chromatin.

Mutagen sensitive 209 (Mus209), more often referred to as Proliferating cell nuclear antigen (PCNA) is a DNA damage-inducible protein that performs essential functions in normal DNA replication, including the resynthesis during nucleotide excision repair of damaged DNA, as an auxiliary factor for DNA polymerases delta and epsilon. DNA polymerase delta is the functional DNA polymerase on the leading strand of the eucaryotic DNA replication fork. The high speed and processivity of replicative DNA polymerases reside in the processivity factor known as PCNA, which has been shown to be a ring-shaped protein. This protein, a sliding clamp, encircles DNA and tethers the DNA polymerase catalytic unit to the DNA template. Mammalian PCNA is loaded onto DNA by a special protein complex known as Replication factor C (RFC). RFC carries out multiple functions: these include the ability to recognize and bind to a DNA primer end and load the ring-shaped PCNA onto DNA in an ATP-dependent reaction. PCNA then tethers the polymerase to the template, allowing processive DNA chain elongation.

Perhaps the most intriguing property of temperature sensitive mutations in the mus209 gene that codes for Drosophila PCNA is suppression of the gene inactivation phenomenon known as position effect variagation (PEV). PEV in essence refers to a chromosomal rearrangement; the placing of a gene near heterochromatin, which leads to the inactivation of that gene in a capricious manner. Heterochromatin is the genetically silent area of a chromosome, a quiet haven in what is otherwise a noisy workzone. Variegation produces a quixotic gene expression; the gene is now turned on, now turned off. Variation in expression is found from fly to fly, and even between different cells in the same fly. This either on or off gene status is passed along from a cell to that cell's offspring. mus209 suppresses the PEV caused by the rearrangement of indicator genes to positions closer to the heterochromatin (Henderson, 1994).

The compaction model of PEV posits that gene inactivation occurs as the variegating gene becomes packaged as heterochromatin. In PEV, chromatin componenets normally restricted to heterochromatin are assumed to spread beyond the euchromatin-heterochromatin boundary, resulting in heterochromatinization of euchromatin and transcriptional inactivation of the variegating gene. To rationalize this model with the experimental results, requires that PCNA be either a component of heterochromatin or be involved somehow in chromatin assembly. It is suggested that suppression of variegation by mus209 mutants could be explained by postulating that an interaction between mutant PCNA and the Drosophila histone assembly machinery is impaired, thereby altering nucleosome positioning at euchromatin-heterochromatin boundaries. Alternatively, PCNA might be involved in regulating the timing of replicon firing or the rate of DNA polymerase progression during S phase, which may influence the transcriptional state of the gene. Thus, in mus209 mutants, a variegating gene might be expressed simply as a consequence of euchromatin-heterochromatin junctions undergoing early replication or altered chromatin assembly (Henderson, 1994 and references).

Recent evidence ties PCNA directly to a Polycomb group protein Cramped (Crm). Polycomb group proteins are involved in global gene silencing in Drosophila. The S-phase-specific nuclear localization of Cramped is reminiscent of the same localization in PCNA. During the first 13 nuclear division cycles, PCNA is present in all interphase nuclei and absent from metaphase chromosomes. Double immunostaining of Crm and PCNA reveals that the appearance and disappearance of the nuclear signals are identically timed during the preblastoderm cycles; by gastrulation, both proteins show overlapping patterns of expression. These two proteins are clearly observed in polytene tissues, and their staining pattens overlap. There is a genetic interaction between crm and mus209, the Drosophila gene encoding PCNA. Thus Crm may be the link between PCNA and Polycomb proteins regulating position effect variagation (Yamamoto, 1997).

Of special interest for an understanding PCNA function are the extensive analyses of the Drosophila mus209 promoter by Matsukage, Yamaguchi and their colleagues. The mus209 promoter contains at least three transcriptional regulatory elements: the URE (upstream regulatory element), DRE (DNA replication-related element), and E2F recognition sites. Three DREs are found in the Drosophila DNA polymerase alpha gene (Yamaguchi, 1996), two in the Drosophila raf gene (Ryu, 1997), and one each is found in the Cyclin A gene (Ohno, 1996) and mus209 (Yamaguchi, 1996). While the role of E2F in cell cycle regulation is fairly well documented , the role of DREF is only now beginning to be undersood. DREF is a novel transcription factor with a basic DNA binding domain and acidic and proline rich domains (Hirose, 1996).

Zerknullt (Zen) has been shown to repress mus209, but this effect is likely to be indirect (Yamaguchi, 1991b). The expression of of the Drosophila DNA polymerase alpha gene is also repressed by zerknullt. The expression of the zen results in reduction of the abundance of mRNA expression directed by mus209 and DNA polymerase alpha promoter fragments and also mRNAs for both mus209 and DNA polymerase alpha. Deletions of the DNA replication-related element (DRE) reveal that the DRE sequences are responsible for repression by Zen protein. zen expressing cells contain lesser amounts of the DRE-binding factor (DREF) than do untransfected or mutant zen-transfected cells. These results suggest that the Zen protein represses expression of DNA replication-related genes by reducing DREF, although the detailed mechanism of the repression remains to be elucidated (Hirose, 1994).

The view of PCNA as a mechanical clamp belies the complexity of PCNA protein interactions and the importance of its regulatory roles. Mammalian PCNA interacts with p21 (Drosophila homolog: Dacapo), an inhibitor of cyclin dependent kinases, suggesting that p21 plays a dual role, affecting both cell cycle regulators and the DNA synthetic machinery. PCNA physically interacts with nucleases involved in DNA repair. PCNA interacts with Gadd45, a protein that stimulates DNA excision repair and inhibits entry of cells into S phase. Thus GADD45 provides a link between the p53-dependent cell cycle checkpoint and PCNA involvement in DNA repair. Transcription of PCNA is also regulated by p53, and PCNA transcription is regulated by E2F downstream of Interleukin 2. Thus the wider view of PCNA regulation and protein interactions is a window into the complex regulatory networks involved in gene silencing, cell cycle progression, DNA synthesis, and repair of DNA damage.


GENE STRUCTURE

Immediately adjacent to, but distrinct from the PCNA gene, is plutonium (plu). The PLU gene product controls DNA replication early in Drosophila development. plu mutant females lay unfertilized eggs that have undergone extensive DNA synthesis. In fertilized embryos from plu mutant mothers, S-phase is uncoupled from mitosis. The gene is expressed only in ovaries and embryos; null alleles are strict maternal effect mutations, and the phenotype of inappropriate DNA replication is the consequence of loss-of-gene function. plu therefore negatively regulates S-phase at a time in early development when commitment to S-phase does not depend on cyclic transcription. plu encodes a protein with two ankyrin-like repeats, including a domain for protein-protein interaction (Axton, 1994).

cDNA clone length - 1.1kb

Bases in 5' UTR - 89

Exons - 2

Bases in 3' UTR - 374


PROTEIN STRUCTURE
Amino Acids - 260

Structural Domains

A protein with an apparent mass of 36 kDa was purified from Drosophila melanogaster embryos using a protocol developed for the purification of proliferating cell nuclear antigen (PCNA) from human 293 cells. The Drosophila protein comigrates with human PCNA on one-dimensional sodium dodecyl sulfate-polyacrylamide gels and cross-reacts with monoclonal anti-rabbit PCNA antibodies. NH2-terminal amino acid sequence analysis reveals that the putative Drosophila PCNA is highly homologous to human PCNA. Of the first 22 amino acids, 16 are found to be identical between the two species, and four of the remaining six are changed conservatively. Results of total amino acid analysis are also consistent with a high degree of similarity between Drosophila PCNA and human PCNA. Functional analysis using the reconstituted simian virus 40 in vitro DNA replication system demonstrates that Drosophila PCNA can substitute, albeit with reduced efficiency, for human PCNA in stimulating simian virus 40 DNA synthesis. Affinity-purified anti-Drosophila PCNA antibodies cross-react with human PCNA and are able to recognize specifically Drosophila PCNA both on crude homogenate immunoblots and by indirect immunofluorescence analysis of proliferating cells in larval tissues in situ (Ng, 1990).

The genomic and cDNA clones for a Drosophila proliferating cell nuclear antigen (PCNA) were isolated and sequenced. The coding sequence for a 260-amino-acid residue polypeptide is interrupted by a single short intron of 60 base pairs (bp); about 70% of the deduced amino acid sequence of the Drosophila PCNA is identical to the rat and human PCNA polypeptides, with conserved unique repeats of leucine in the C-terminal region. The highly conserved sequence between residues 61 and 80 shows an alpha-helix-turn-alpha-helix motif: this is a putative DNA-binding domain found in several proteins. Genomic Southern blot hybridization analysis indicates the presence of a single gene for PCNA per genome (Yamaguchi, 1990).


Proliferating cell nuclear antigen: Evolutionary Homologs | Regulation | Developmental Biology | Effects of Mutation | References

date revised: 1 Nov 97

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