DNA polymerase-associated lectin (DPAL) and its binding to the galactose-containing glycoconjugate of the replication complex.

Abstract

The highly purified DNA Pol-alpha from rat prostate tumor (PA-3) and human neuroblastoma (IMR-32) cells appeared to be inhibited by Ricin (RCA-II), and Con-A. Loss of activity (40 to 60%) of a specific form of DNA polymerase from IMR-32 was observed when the cells were treated with tunicamycin [Bhattacharya, P. and Basu, S. (1982) Proc. Natl. Acad. Sci., USA 79:1488-1492]. Binding of ConA and RCA to human recombinant DNA polymerase-alpha showed a specific labile site in the N-terminus [Hsi et al.. (1990) Nucleic Acid Res. 18:6231-6237]. The catalytic polypeptide, DNA polymerase-alpha of eukaryotic origin, was isolated from developing tissues or cultured cells as a family of 180 to 120 kDa polypeptides, perhaps derived from a single primary structure. Immunoblot analysis with a monoclonal antibody (SJK-237-71) indicated that the lower molecular weight polypeptides resulted from either proteolytic cleavage of post-translational modification after specific cleavages. Present results suggest DNA polymerase-alpha from embryonic chicken brain (ECB) contains an alpha-galactose-binding subunit which may be involved in developmental regulation of the enzyme. It was shown before that the catalytic subunit of DNA polymerase-alpha reduces from 186 kDa in 11-day-old ECB to 120 kDa in 19-day-old ECB [Ray, S. et al. Cell Growth and Differentiation 2:567-573] by the treatment with methyl-alpha-galactose. The low molecular weight DNA polymerase activity (120 kDa) can be reconstituted to high molecular weight (Mr = 186 kDa) with an alpha-galactose binding, 56kDa lectin-like protein. Polyclonal antibodies raised against the purified lectin were able to precipitate DNA. Pol-alpha as determined by immunostaining with the polymerase-alpha-specific monoclonal antibody SJK 132-20, suggesting this is a DNA polymerase associated-lectin (DPAL). RCA-II and GS-I-Sepharose 4B chromatographies resulted in significant purification of DNA-alpha and a complete separation of polymerase complex and primase.

• Authors: T J Kelley, T St Amand, J M Groll, S Ray, S Basu
• Journal: Bioscience reports (Biosci Rep) Vol. 19 Issue 5 Pg. 433-47 (Oct 1999) ISSN: 0144-8463 [Print] England
• PMID: 10763811 (Publication Type: Journal Article, Research Support, U.S. Gov't, P.H.S.)

Chemical References

• Glycoconjugates
• Lectins
• Methylgalactosides
• RCA II
• Toxins, Biological
• Concanavalin A
• methyl beta-galactoside
• DNA Primase
• DNA Polymerase I
• Galactose

Topics

• Animals
• Catalytic Domain
• Chick Embryo
• Chromatography, High Pressure Liquid
• Concanavalin A (chemistry)
• DNA Polymerase I (chemistry)
• DNA Primase (chemistry)
• DNA Replication
• Galactose (chemistry)
• Glycoconjugates (chemistry)
• Humans
• Lectins (chemistry)
• Methylgalactosides (chemistry)
• Molecular Weight
• Precipitin Tests
• Protein Binding
• Rats
• Toxins, Biological (chemistry)
• Tumor Cells, Cultured