Intracellular localization and metabolism of DNA polymerase α in human cells visualized with monoclonal antibody

Nakamura, Hiromu; Morita, Toshiteru; Masaki, Shiego; Yoshida, Shonen

doi:10.1016/0014-4827(84)90362-8

Cited by 116 publications

(43 citation statements)

References 13 publications

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“…Like the observations of pol ⑀ described here, the human replicative polymerase, pol ␣, has been reported to be localized to nuclear foci in S phase that colocalize with sites of DNA synthesis (44,49,58). Although the localization of pol ␦ has not been reported, it is also assumed to be a component of replication foci as its processivity factor, PCNA, colocalizes to replication foci (51,56).…”

Section: Discussionsupporting

confidence: 75%

Human DNA Polymerase ε Colocalizes with Proliferating Cell Nuclear Antigen and DNA Replication Late, but Not Early, in S Phase

Fuss

Linn

2002

Journal of Biological Chemistry

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DNA polymerase ⑀ (pol ⑀) has been implicated in DNA replication, DNA repair, and cell cycle control, but its precise roles are unclear. When the subcellular localization of human pol ⑀ was examined by indirect immunofluorescence, pol ⑀ appeared in discrete nuclear foci that colocalized with proliferating cell nuclear antigen (PCNA) foci and sites of DNA synthesis only late in S phase. Early in S phase, pol ⑀ foci were adjacent to PCNA foci. In contrast to PCNA foci that were only present in S phase, pol ⑀ foci were present throughout mitosis and the G 1 phase of cycling cells. It is hypothesized from these observations that pol ⑀ and PCNA have separate but associated functions early in S phase and that pol ⑀ participates with PCNA in DNA replication late in S phase.Although as many as 13 DNA polymerases are known to exist in eukaryotes, only 3 are thought to be involved in chromosomal replication; they are DNA polymerases (pol) 1 ␣, ␦, and ⑀. pol ␣ is clearly involved in the synthesis and extension of RNA primers during DNA replication initiation (reviewed in Ref. 1), whereas pol ␦ is responsible for elongation, as most clearly demonstrated in SV40 DNA replication (2, 3). Although pol ⑀ is not required for SV40 replication (4, 5), it does appear to have a role in chromosomal DNA replication (5-10).DNA pol ⑀ is well conserved in sequence and subunit composition among eukaryotes. Human pol ⑀ has four subunits, a large catalytic subunit, p261, and three associated subunits, p59, p12, and p17 (11,12). Likewise, Saccharomyces cerevisiae pol ⑀ has a catalytic subunit of 256 kDa, POL2, and has three associated subunits encoded by DPB2, DPB3, DPB4 (8, 13-15).Both catalytic subunits are divided into two domains linked by a protease-sensitive region, an N-terminal domain containing polymerase and exonuclease motifs and a 120-kDa C-terminal domain. The S. cerevisiae and human catalytic subunits are 39% identical overall and have 63% identity in the N-terminal domain (16). p59 and DPB2p have 26% overall identity (17), whereas p17 and p12 have 36.5 and 34% identity to Dpb4p and Dpb3p, respectively (12). Given the conservation of pol ⑀ sequence and subunit structure between yeast and humans, it is likely that their functions are also largely conserved.A role for pol ⑀ in chromosomal DNA replication has been established in higher eukaryotes and in yeast. The elongation step of DNA replication is impaired in Xenopus laevis egg extracts immunodepleted of pol ⑀ (6). Additionally, using a polymerase trap technique that tags a polymerase with its DNA product, Zlotkin et al. (5) find that pol ⑀ from monkey kidney cells associates with nascent chromosomal DNA but not nascent SV40 DNA. Likewise, a neutralizing antibody against human pol ⑀ inhibits chromosomal replication when microinjected into human fibroblasts but fails to inhibit SV40 DNA replication in vitro (7). In S. cerevisiae, disruptions of POL2 are lethal and result in the terminal dumbbell morphology characteristic of a defect in DNA replication (8). When shifted to the no...

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Section: Discussionsupporting

confidence: 75%

Human DNA Polymerase ε Colocalizes with Proliferating Cell Nuclear Antigen and DNA Replication Late, but Not Early, in S Phase

Fuss

Linn

2002

Journal of Biological Chemistry

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“…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).…”

Section: Immunoblottingmentioning

confidence: 58%

“…On the other hand, Nakamura et al (1984) have also reported that they established a mouse hybridoma clone secreting an antibody against calf thymus O10S DNA polymerase a, which cross-reacted with human a-enzyme, and that indirect immunofluorescence microscopy with MAb against DNA polymerase a revealed the intranuclear localisation of DNA polymerase a in G1, S and G2 phases of transformed human cells (Nakamura et al, 1984).…”

Section: Immunoblottingmentioning

confidence: 99%

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

Sugawara

Uchino²,

Morishita³

et al. 1989

Br J Cancer

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Summary We have successfully established one murine hybridoma that secretes a monoclonal antibody specific for the 77,000 subunit of human DNA polymerase a. The results of immunochemical studies, using We have reported the preparation and characterisation of two stable murine hybridomas that secrete monoclonal antibodies (MAb) (HDR-854-E4 and HDR-863-A5) specific for HeLa cell DNA polymerase cx, one of which (HDR-854-E4) recognises the 77-kDa subunit of DNA polymerase a (Yagura et al., 1987). Binding of HDR-854-E4 MAb also interferes with the action of the primase subunit (Yagura et al., 1987).In this paper, we describe results obtained in our initial effort to apply HDR-854-E4 MAb to human cultured cell lines and various human tissues for the immunocytochemical localisation of the DNA polymerase c subunit by light and electron microscopy and to biopsies of the thyroid and gastrointestinal (GI) tract. Materials and methods Cells and tissuesVarious cell lines including HeLa (human uterine cervix cancer), SUIT-2 (human pancreatic cancer), Marcus (human glioblastoma), TIG (human fibroblast), KATO III (gastric cancer), CEM (T-cell lymphoma), COLO 205 (colonic cancer), Daudi (Burkitt lymphoma), K-562 (myelogenous leukaemia), BALL-1 (acute B-cell leukaemia), NALM-6 (acute leukaemia), PANC-1 (pancreatic cancer), KB (oral epidermoid cancer), HT-1080 (human fibrosarcoma), KD (human lip fibroblast), MRC-9 (human fetal lung fibroblast), BeWo (human choriocarcinoma) and U-937 (human histiocytic lymphoma) were obtained from the Japanese Cell Resources Bank (JCRB), Tokyo, Japan. Science, University of Tokyo. For immunostaining the tissue was snap-frozen and stored until use at -70°C. AntibodiesThe generation and specificity of the monoclonal antibody HDR-854-E4 have been described in detail elsewhere (Yagura et al., 1987). SJK 132-20 MAb was kindly supplied by Professor D. Korn, Department of Pathology, Stanford University School of Medicine, and its characterisation has been decribed elsewhere . We used this MAb for comparison with HDR-854-E4. Biotinylated horse anti-mouse IgG antibody and avidin-biotin-peroxidase complex (ABC-PO) reagent were purchased from Vector Labs. (Burlingame, CA, USA) (Hsu et al., 1981 IgG Ab was applied and incubated at RT for 30 min. After careful washing with PBS, the specimens were colorated with 4.5 mg diaminobenzidine (DAB) and 10 Ml of 30% H202 in 20 ml PBS after a 30-min incubation with ABC-PO solution (1:100-diluted).

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“…(1984) specifically describe results obtained on human lymphocyte mitotic chromosomes. In all cases, it seems that the abovecited immunoreaction occurs in the cytoplasm but not on chromatin (Nakamura et at., 1984;Stokke et at., 1991). Our data, on the contrary, show intense immunoreaction in human nuclei and mitotic chromosomes after McAb staining.…”

Section: Discussionmentioning

confidence: 99%

“…This apparent contradiction might be explained by the fact that the above-cited authors employed procedures that were different compared with those used in our experiments. For instance, a different McAb was employed by Nakamura et al (1984), whereas Stokke et a!.…”

Section: Discussionmentioning

confidence: 99%

On the presence of DNA polymerase α in human lymphocyte nuclei and chromosomes

et al. 1996

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Experiments were carried out to correlate the cytological localization of DNA polymerase with the presence of its specific mRNA in human lymphocytes studied at different times after phytohaemagglutinin stimulation. Our data indicated that in resting cells it is not possible to detect DNA polymerase c protein or mRNA by Northern hybridization. By contrast, in stimulated cells the detection of mRNA specific for DNA polymerase c synthesis is possible after 16 h phytohaemagglutin stimulation, whereas immunolocalization is possible after only 4 h stimulation. Observation of cytological preparations from cells stimulated for times long enough to obtain mitoses surprisingly showed an intense immunoreaction in mitotic chromosomes treated with monoclonal antibodies to DNA polymerase .

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Intracellular localization and metabolism of DNA polymerase α in human cells visualized with monoclonal antibody

Cited by 116 publications

References 13 publications

Human DNA Polymerase ε Colocalizes with Proliferating Cell Nuclear Antigen and DNA Replication Late, but Not Early, in S Phase

Human DNA Polymerase ε Colocalizes with Proliferating Cell Nuclear Antigen and DNA Replication Late, but Not Early, in S Phase

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

On the presence of DNA polymerase α in human lymphocyte nuclei and chromosomes

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