Intracellular localization of DNA polymerase alpha.

Abstract

Immunoglobulin (IgG) and the F(ab')2 fragment of IgG were prepared from serum of a rabbit immunized with purified calf thymus DNA polymerase a (deoxynucleosidetriphosphate:DNA deoxynucleotidyltransferase, EC 2.7.7.7). An indirect immunofluorescent method based on these reagents was used to detect the intracellular localization of DNA polymerase a in primary fetal bovine fibroblasts. The results show that the bulk of DNA polymerase a is located in the perinuclear region of the cytoplasm. Immunofluorescent staining ofcytoplast and FicollPaque gradient-purified karyoplast fragments resulting from cytochalasin enucleation show the presence of DNA polymerase a in cytoplasts and the virtual absence of the enzyme in the nucleus of the karyoplast itself. The implication of this unusual intracellular location for DNA polymerase a is discussed.The original description of DNA polymerase (deoxynucleosidetriphosphate:DNA deoxynucleotidyltransferase, EC 2.7.7.7) in eukaryotic systems (1, 2) noted the presence of the major amount of its activity in the cytoplasmic fraction of buffered sucrose homogenates, with a minor amount of activity in the nucleus. This finding has been confirmed by other early investigators (3). A report suggesting transport of activity to the nucleus during S phase has been published also (4). Subsequent work with nonaqueous solvents (5, 6) has suggested a nuclear localization. Use of cytochalasin B to produce cytoplasts and karyoplasts also has been interpreted to prove a nuclear localization of DNA polymerase a (7). Biochemical studies routinely use a cytoplasmic fraction for isolation of DNA polymerase a'.DNA polymerase 18 has been found to be nuclear and cytoplasmic (8, 9) and DNA polymerase y has been localized in mitochondria (10,11).Because all of these studies have required breakage of cells, the arguments have centered on how the cells were disrupted and the quantitative analysis of the result. This matter could be settled by demonstrating localization of the enzyme in wholecell preparations. We Antibody to DNA Polymerase a. The antibody used for this study was a monospecific antibody against DNA polymerase a, prepared by immunizing a rabbit with calf thymus 'DNA polymerase a. Immunoglobulin (IgG) and F(ab')2 fragment of IgG were prepared from the immune serum. The preparation and characterization of this antibody have been described (12).Immunofluorescence. An indirect immunofluorescent technique was used to test for the presence of DNA polymerase a. Cells growing on cover slips, cytocentrifuge preparations of purified karyoplasts, and re-adhered cytoplasts were washed three times in medium F-10 without serum, fixed in absolute methanol for 10 min at 40C, and air dried. The IgG or F(ab')2 fragment of rabbit anti-calf thymus DNA polymerase a or preimmune IgG (diluted to 100-400 Ag/ml in phosphate-buffered saline) was used in the primary incubation for 30 min at room temperature. The secondary antibody was the fluorescein isothiocyanate-conjugated F(ab')2 fragment ofgoat anti-rabbit Ig...

Intracellular localisation of a subunit of human DNA polymerase α affecting primase activity recognised by monoclonal antibody (HDR-854-E4) and its application to distinction between proliferative and non-proliferative lesions

“…Thus, we believe that our results are reliable and reproducible. it is not fruitful to speculate here about the possible reasons for the disparity between the immunocytochemical results reported in this paper and those obtained by Brown et al (1981), Bensch et al (1982) and Nakamura et al (1984). However, we wish to emphasise that successful application of the cytochemical methods described in this paper is critically dependent on the fixation protocol (Loke et al, 1988).…”

“…Brown et al (1981) have described the results of immunohistochemical studies that appeared to demonstrate the essentially exclusive cytoplasmic localisation of DNA polymerase a in fixed whole cell preparations of two lines of cultured bovine cells, as well as the absence of detectable polymerase a antigens from gradient-purified karyoplast fractions. They used a polyclonal rabbit antiserum that had been raised against an incompletely purified calf thymus polymerase a fraction, which was claimed to be monospecific, and an immunofluorescence detection system, employing washed monolayer cell cultures that had been fixed in absolute methanol for 10 min at 4°C (Brown et al, 1981). Bensch et al (1982) have reported that the immunoperoxidase reaction product corresponding to human polymerase a exhibits a diffuse pattern of distribution within the nucleoplasm, whereas nucleoli are clearly negative.…”

Delayed and reduced cell replication and diminishing levels of DNA polymerase‐α in regenerating liver of aging mice

“…Numerous authors have provided evidence to show that polymerase-a resides within the nucleus of animal cells (Lynch et al, 1975;Foster and Gurney, 1976;Fox et al, 1980;Herrick et al, 1976). Other investigators countered with observations suggesting that polymerase-a is localized mainly in the cytoplasm (Byrnes et al, 1974;Tarrago-Litvsk et al, 1979;Brown et al, 1981). Third, it has been argued by several researchers that polymerase-a may migrate from cytoplasm to nucleus as cells traverse from the G1 to the S phase of the cell cycle (Loeb and Fansler, 1970;Fansler and Loeb, 1972;Hobart and Infante, 1980;Reddi and Pardee, 1980).…”

Association of ubiquitin-activating enzyme with HeLa cell chromosomes during mitosis.

“…To demonstrate that the immunoreactive material was El, three conditions were met (18): (i) affinity purification was used to eliminate contaminating antibodies from the polyclonal serum, (ii) low concentrations of anti-El-specific antibody (0.5 pug/ml) were used to prevent low-affinity cross reactions, and (iii) Western (19,20).…”