Probable involvement of a glycoconjugate in IMR-32 DNA synthesis: decrease of DNA polymerase alpha 2 activity after tunicamycin treatment.

Abstract

Multiple forms of DNA polymerase a activity (al, a2, and a3) from human neuroblastoma IMR-32 cells untreated or treated with tunicamycin (3 ,ug/ml) were separated by DEAE-cellulose column chromatography. Loss of40-60% ofDNA polymerase a2 activity was observed in tunicamycin-treated cells. Ricin IB, a subunit of intact ricin (Mr, 64,000), was found to be a specific inhibitor of DNA polymerase a2 isolated from control IMR-32 cells. However We report here that tunicamycin (19), which blocks the synthesis of N-acetylglucosaminylpyrophosphorylpolyisoprenol and thereby inhibits the glycosylation of protein (20-23), specifically decreases the activity of DNA polymerase a2.EXPERIMENTAL PROCEDURE Nucleotides and Polynucleotides. 3H-Labeled deoxynucleotides were purchased from New England Nuclear and ICN. Synthetic polynucleotide templates and oligonucleotide primers were obtained from either Miles Laboratories or P-L Biochemicals. Template-primer hybridization was performed according to Wickremasinghe (24). A mixture of template and primer (20:1) in 10 mM NaCl was heated for 10 min at 550C, cooled at room temperature, and stored at -20'C. In the text that follows, subscripts denote chain lengths of polynucleotide primers. Tunicamycin (lot NCI-2) was a gift from John D. Douros (National Cancer Institute).Cell Culture. Cultures of human neuroblastoma IMR-32 cells (a gift from S. Brooks) were grown in 250-ml (75-cm2) Falcon plastic flasks containing 15-20 ml of minimal essential medium supplemented with 10% fetal calf serum (GIBCO) (14). The medium also contained 100 units of penicillin/streptomycin (GIBCO) per ml to prevent microbial growth. Incubation was carried out in a humidified atmosphere of 97.5% air/2.5% CO2. The medium was changed twice weekly, and cells were subcultured upon reaching confluence. The experimental cells were maintained in minimal essential medium containing tunicamycin (3 ,ug/ml) for 40 hr to obtain maximal inhibition of DNA synthesis. The cells were harvested with 5.0 ml of phosphate-buffered saline (pH 7.2) containing 0.1% EDTA per T flask. After pelleting by centrifugation, the cells were suspended in 2-3 vol of 0.32 M sucrose and stored at -200C.Separation of DNA Polymerases on Sucrose Density Gradients. Sucrose density gradients were prepared by using 5-20% sucrose in 10 mM Tris HCl, pH 8.0/100 mM KCVl1 mM mercaptoethanol as described (14). Aliquots (0.3-0.4 ml) ofsonicated IMR-32 supernatant were layered on the gradients (4.5 ml) and centrifuged for 16 hr at 149,000 X g in a Beckman SW 50.1 rotor. Fractions (0.25 ml) were collected from the bottom with a peristaltic pump. DNA polymerase a and f8 activities were found in fractions 5 and 14, respectively.Isolation of Multiple Forms of DNA Polymerase a by DEAE-Cellulose Column Chromatography. The soluble supernatants from tunicamycin-treated or control samples were loaded on separate, identical DEAE-cellulose columns (2 X 14 cm) that had been equilibrated with 100 ml of 50 mM potassium phosphate, pH 7.6/1 mM mercaptoethanol. The proteins...

Fundamental Aspects of DNA Replication

“…Reportedly, several lectins such as Concanavalin A bound human catalytic subunit of pol-α (p180), and that ricin from castor beans bound and inhibited pol-α, suggesting that the p180 subunit has carbohydrate chains recognized by these lectins (Bhattacharya et al, 1979;Hsi et al, 1990). Because tunicamycin, which inhibits the glycosylation of protein, has been reported to decrease the activity of pol-α (Bhattacharya & Basu, 1982), it is suggested that the carbohydrate chain on pol-α is important for its activity. However, the detailed roles of carbohydrate chains on p180 subunit have not been elucidated to date.…”

Replication of single-stranded DNA templates by primase-polymerase complexes of the yeast,Saccharomyces cerevisiae

“…However, this assay is not capable of appraising many in vivo actions of DNA polymerase I, such as rate, processivity, etc. Various forms of DNA polymerase a (or I) have been isolated from a wide variety of eukaryotic cells (3)(4)(5)(6)(7)(8)(9)(10)(11)(12). The complexity of structural variations among different preparations may have been, in part, due to the fragility of the multiprotein polymerase assembly, proteolytic degradation, and lack of suitable assays capable of assessing the in vivo actions of a multifunctional polymerase.…”

Function of DNA Polymerase α in a Replication Fork and its Putative Roles in Genomic Stability and Eukaryotic Evolution

“…Reportedly, several lectins such as Concanavalin A bound human catalytic subunit of pol-(p180), and that ricin from castor beans bound and inhibited pol-, suggesting that the p180 subunit has carbohydrate chains recognized by these lectins (Bhattacharya et al, 1979;Hsi et al, 1990). Because tunicamycin, which inhibits the glycosylation of protein, has been reported to decrease the activity of pol- (Bhattacharya & Basu, 1982), it is suggested that the carbohydrate chain on pol-is important for its activity. However, the detailed roles of carbohydrate chains on p180 subunit have not been elucidated to date.…”