Proliferating cell nuclear antigen



The PCNA mRNA is detected at high levels in adult ovaries, unfertilized eggs, and early embryos and at low levels in the other developmental stages. The abundance of the mRNA in the adult ovary suggests transfer of the maternal mRNA into the ooplasm. In fact, the PCNA transcripts accumulate in the unfertilized eggs laid by virgin females. Thus, the transcripts in the early embryonic stages are contributed mainly by maternal gene expression (Yamaguchi, 1990).

An immunocytochemical method using a specific antibody was employed to detect the proliferating cell nuclear antigen (PCNA) in Drosophila embryos during the first 13 nuclear division cycles. Strong nuclear staining with the anti-PCNA antibody is observed at interphase throughout 13 cycles. Metaphase chromosomes are not stained throughout these cycles. The chromosomal (nuclear) staining reappears at anaphase until cycle 10 and at telophase in cycle 11. During cycles 12 and 13, nuclear staining is detected exclusively at interphase. Relatively uniform staining of syncytial cytoplasm is observed throughout mitotic phases until cycle 9. In the following cycles, strong staining in both the central yolk mass and the cortical layer of cytoplasm is detected at metaphase and telophase. During interphase of cycles later than the 9th, staining in the central yolk mass gets much fainter; staining in the cortical cytoplasm completely disappears. These results suggest that the PCNA dissociates from chromosomes at metaphase; in later mitotic phases, it is transported from the syncytial cytoplasm into nuclei to participate in formation of the active DNA-replication enzyme complexes (Yamaguchi, 1991a).

Proliferating cell nuclear antigen (PCNA) is present throughout the mitotic cycle. During postblastoderm cell division cycles strong nuclear staining observed during interphase disappears at prophase. Staining remains nil throughout the remaining mitotic (M) phases and reappears in nuclei at the next interphase. Disappearance of the staining signal depends on string function and is coupled with the onset of mitosis. When cells in embryos are arrested at M phase following treatment with TN-16 or Vinblastine, the staining signal with the anti-PCNA antibody is lost. However, the level of PCNA protein in M phase-arrested embryos revealed by western blots does not decrease. Therefore, the disappearance of staining signals is apparently due to reorganization of PCNA protein in the multiprotein complex in nuclei, rendering it inaccessible to the antibody, rather than to the degradation of PCNA protein. Cultured Drosophila cells stain strongly with the anti-PCNA antibody during both interphase and the M phase (Yamaguchi, 1995c).

Effects of Mutation or Deletion

There is a genetic interaction between cramped(crm) and mus209, the Drosophila gene encoding PCNA. Drosophila cramped of can be classified as a Polycomb-group (Pc-G) gene. crm mutants exhibit typical Pc-G mutant phenotypes, reminiscent of ectopic homeotic gene expression, with additional sex comb teeth found on mesothoracic and metathoracic legs, and proximodistal transformations of the tarsal segments. crm encodes an 693 amino acids protein, with no significant homology to known proteins. Polyclonal antibodies raised against bacterially expressed truncated Crm protein were used to show that the crm gene product is localized to the nucleus during embryogenesis. This nuclear localization appears to be restricted to S-phase nuclei, as Crm immunostaining disappears at mitosis. This cell-cycle-dependent staining pattern is identical to that of proliferating cell nuclear antigen (PCNA). Crm and PCNA proteins are co-localized in salivary gland polytene nuclei. Together, these data suggest that these two proteins are involved in a common regulatory pathway and highlight possible interactions between Pc-G-mediated silencing and DNA replication in Drosophila (Yamamoto, 1997).

The mus209B1 mutant of Drosophila exhibits a complex pleiotropy of temperature-sensitive (ts) lethality, hypersensitivity to DNA-damaging agents such as ionizing radiation and methyl methanesulfonate, suppression of position-effect variegation (PEV), and female sterility. Although a small number of adults die at each larval stage, and still others pupate but die as pharate adults prior to eclosion, most puparia come to encase an amorphous mass of disintegrating larval tissue without discernible adult structures. mus209 encodes PCNA, an indispensable component of the DNA replication apparatus. This suggests that alterations to chromosome replication may underlie that pleiotropy. Nine lethal mutations, three of them ts, genetically define the Pcna locus. Temperature shift studies reveal that the vital function of PCNA is required throughout virtually all stages of fly development, and that maternally encoded PCNA is essential for embryogenesis. All three ts mutants strongly suppress PEV, which suggests a role for PCNA in chromatin assembly or modification (Henderson, 1994).

PCNA functions in DNA replication as a processivity factor for polymerases delta and epsilon, and in multiple DNA repair processes. Two temperature-sensitive lethal alleles (mus209B1 and mus2092735) of the Drosophila PCNA gene are described that, at temperatures permissive for growth, result in hypersensitivity to DNA-damaging agents, suppression of position-effect variegation, and female sterility in which ovaries are underdeveloped and do not produce eggs. The sterility of mus209B1 is partly due to a failure of germ-line cells to proliferate. Strikingly, mus209B1 and mus2092735 interact to restore partial fertility to heteroallelic females, revealing additional roles for PCNA in ovarian development, meiotic recombination, and embryogenesis. Although mus209B1 and mus2092735 homozygotes are each defective in repair of transposase-induced DNA double-strand breaks in somatic cells, this defect is substantially reversed in the heteroallelic mutant genotype. These novel mutations map to adjacent sites on the three-dimensional structure of PCNA, which was unexpected in the context of this observed interallelic complementation. These mutations reveal new relationships between the structure and function of PCNA (Henderson, 2000)

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


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