Differential inhibition of multiple forms of DNA polymerase alpha from IMR-32 human neuroblastoma cells.

Bhattacharya, Prabir; Simet, Ira M.; Basu, Subhash

doi:10.1073/pnas.78.5.2683

Cited by 14 publications

(10 citation statements)

References 35 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…As mentioned above, hemin has been shown to inhibit retroviral RT and other DNA polymerases (6,35). We have also found that hemin and the related porphyrin compounds could inhibit the purified Moloney murine leuke-mia virus RT and the Klenow fragment of the Escherichia coli DNA polymerase I with a potency only slightly less than their inhibitory effects on the HBV and DHBV RT (Li and Hu, unpublished).…”

Section: Discussionmentioning

confidence: 71%

Inhibition of Hepadnavirus Reverse Transcriptase-ε RNA Interaction by Porphyrin Compounds

2008

J Virol

View full text Add to dashboard Cite

The hepatitis B virus (HBV) reverse transcriptase (RT) plays a multitude of fundamental roles in the viral life cycle and is the key target in the development of anti-HBV chemotherapy. We report here that the endogenous small molecule iron protoporphyrin IX (hemin) and several related porphyrin compounds potently blocked a critical RT interaction with the viral RNA packaging signal/origin of replication, called . As RTinteraction is essential for the initiation of viral reverse transcription, which is primed by RT itself (protein priming), the porphyrin compounds dramatically suppressed the protein-priming reaction. Further studies demonstrated that these compounds could target the unique N-terminal domain of the RT protein, the so-called terminal protein. Hemin and related porphyrin compounds thus represent a novel class of agents that can block HBV RT functions through a mechanism and target that are completely distinct from those of existing anti-HBV drugs.Hepatitis B virus (HBV) belongs to the family Hepadnaviridae, a group of small, hepatotropic DNA viruses that also includes related animal viruses, such as the duck HBV (DHBV) and the woodchuck hepatitis virus (31). Chronic HBV infection remains a major public health problem worldwide, with over 350 million chronic HBV carriers who are at serious risk of developing liver cirrhosis and hepatocellular carcinoma (5,7,12). At present, the immunomodulatory and antiviral cytokine alpha interferon and several nucleoside analogs represent two distinct classes of therapies for chronic HBV infections (14). Unfortunately, alpha interferon therapy induces a sustained antiviral response in only 20 to 30% of patients and is fraught with adverse reactions. In the short term, nucleoside analogs exhibit a potent inhibitory effect on HBV DNA synthesis. However, long-term treatment with nucleoside analogs is required to maintain viral suppression, which leads to the development of drug-resistant HBV variants (13,26). Therefore, the current inventory of therapeutics against HBV infections is inadequate, and novel anti-HBV therapy needs to be developed.All hepadnaviruses replicate the DNA genome through an RNA intermediate (the pregenomic RNA [pgRNA]) by reverse transcription (31, 33). The virally encoded reverse transcriptase (RT) protein carries the two essential enzymatic activities required for the conversion of pgRNA to the DNA copy, i.e., DNA polymerase activity and RNase H activity (18). In contrast to retroviral RTs, the hepadnavirus RT initiates viral DNA synthesis via a novel protein-priming mechanism, whereby the RT protein itself serves a dual role as a protein primer and as the polymerase. As an obligate template for the protein-priming action, a short (ca. 60-nucleotide) viral RNA structure termed ε, located at the 5Ј end of pgRNA, is specifically recognized by the RT protein to form a stable ribonucleoprotein (RNP) complex (28, 38). Using an internal bulge located on the ε stem-loop structure as a template and a specific tyrosine residue of the RT protein as a pr...

show abstract

Section: Discussionmentioning

confidence: 71%

Inhibition of Hepadnavirus Reverse Transcriptase-ε RNA Interaction by Porphyrin Compounds

2008

J Virol

View full text Add to dashboard Cite

show abstract

“…The primase subunit, closely associated with DNA polymerase a, initiates RNA primers before any DNA chain initiation in mouse tumor cells (4,5), Drosophila (6,7), Xenopus laevis (8), human KB cells (9), yeast (10)(11)(12)(13)(14), calf thymus (15,16), human IMR-32 neuroblastoma cells (17), and embryonic chicken brain (54). In addition to primase activity, multiple forms of DNA polymerases (18)(19)(20)(21)(22)(23)(24) from various eukaryotic sources appear to contain specific proteins (25)(26)(27)(28). The DNA polymerase a isolated from different animal sources (25) appears to be homogeneous on native polyacrylamide gels.…”

mentioning

confidence: 99%

Stimulation of human neuroblastoma DNA polymerase alpha and primase activities by a protein factor isolated from rat liver chromatin.

Takada¹,

Torres-Rosado²,

Ray³

et al. 1986

Proc. Natl. Acad. Sci. U.S.A.

Self Cite

View full text Add to dashboard Cite

Nuclear protein factor type 1 (NPF-1) thatsimulates IMR-32 primase-associated DNA polymerase a, and a2 activities has been purified from a high-salt extract of liver chromatin from 6-month-old rats. The final purified factor lacks DNA polymerase a, RNA polymerase, and DNA-unwinding or topoisomerase type I activities. The stimulatory activity is destroyed by trypsin (60 min at 37C), DNase II (60 min at 370C), and heat treatment (2 min at 68C). The '2I-labeled NPF-1 does not bind to activated calf thymus DNA or poly(dC). However, it forms a ternary complex with DNA in the presence of DNA polymerase a-primase complex (a, and a2). The ternary complex sediments on sucrose density gradient as a heavier band (11S). The NPF-1 also stimulates (2.5-fold) primase-catalyzed incorporation of GMP and dGMP from the corresponding triphosphates on poly(dC) template even in the presence of a high concentration of a-amanitin (400 ,tg/ml). 8.0/1 mM EDTA/6 mM KCI/2 mM MgCl2/1 mM 2-mercaptoethanol/0.1 mM phenylmethylsulfonyl fluoride/0.1% polyethylene glycol (PEG; 8 kDa) using a glass-Teflon homogenizer. The homogenate was centrifuged at 100,000 x g for 1 hr. The supernatant (1.5 ml) was applied to a DE 23 column (1.5 x 10 cm) equilibrated with 50 mM potassium phosphate, pH 7.6/1 mM MgCl2/1 mM 2-mercaptoethanol/0.1% PEG/ 0.1 mM phenylmethylsulfonyl fluoride (buffer C). After

show abstract

“…The existence of multiple forms of DNA polymerase a activity in normal and tumor cells has been well established (1)(2)(3)(4)(5)(6)(7)(8)(9)(10)(11)(12), but it is not yet clear whether these multiple forms arise from a single native protein. However, studies with two highly purified species of DNA polymerase a from mouse myeloma cells have indicated the presence of two different subunits that failed to show any sequence homology on tryptic peptide maps (8).…”

mentioning

confidence: 99%

“…In contrast, Hesslewood et al (6) have shown that one form of rat liver DNA polymerase a activity could be converted to a second form by exposure to mild urea treatment. Recently, we have shown the presence of three forms of DNA polymerase a activity in cultured human neuroblastoma IMR-32 cells (12,13). They all sediment at approximately 7 S but have different template-primer preferences and heat sensitivities (12).…”

mentioning

confidence: 99%

See 1 more Smart Citation

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

Bhattacharya¹,

Basu²

1982

Proc. Natl. Acad. Sci. U.S.A.

Self Cite

View full text Add to dashboard Cite

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...

show abstract

Differential inhibition of multiple forms of DNA polymerase alpha from IMR-32 human neuroblastoma cells.

Cited by 14 publications

References 35 publications

Inhibition of Hepadnavirus Reverse Transcriptase-ε RNA Interaction by Porphyrin Compounds

Inhibition of Hepadnavirus Reverse Transcriptase-ε RNA Interaction by Porphyrin Compounds

Stimulation of human neuroblastoma DNA polymerase alpha and primase activities by a protein factor isolated from rat liver chromatin.

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

Contact Info

Product

Resources

About