DNA Polymerase-Associated Lectin (DPAL) and Its Binding to the Galactose-Containing Glycoconjugate of the Replication Complex |
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Authors: | Thomas J. Kelley Tara St. Amand Jeremy M. Groll Satyajit Ray Subhash Basu |
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Affiliation: | (1) Department of Pediatrics, BRB-836;, CaseWestern Reserve University, 10900 Euclid Ave., Cleveland, OH, 44106;(2) Department of Chemistry and Biochemistry, University of Notre Dame, Notre Dame, IN, 46556;(3) Scripgen Pharmaceutical, 610 Lincoln St., Waltham, MA, 02154 |
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Abstract: | The highly purified DNA Pol- 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., USA79:1488–1492]. Binding of ConA and RCA to human recombinant DNA polymerase- showed a specific labile site in the N-terminus [Hsi et al.. (1990) Nucleic Acid Res.18:6231–6237].The catalytic polypeptide, DNA polymerase- 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- from embryonic chicken brain (ECB) contains an -galactose-binding subunit which may be involved in developmental regulation of the enzyme. It was shown before that the catalytic subunit of DNA polymerase- reduces from 186 kDa in 11-day-old ECB to 120 kDa in 19-day-old ECB [Ray, S. et al. Cell Growth and Differentiation2:567–573] by the treatment with methyl--galactose. The low molecular weight DNA polymerase activity (120 kDa) can be reconstituted to high molecular weight (Mr = 186 kDa) with an -galactose binding, 56 kDa lectin-like protein. Polyclonal antibodies raised against the purified lectin were able to precipitate DNA.Pol- as determined by immunostaining with the polymerase--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- and a complete separation of polymerase complex and primase. |
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Keywords: | Brain DNA Polymerase- /content/p5826751m5u321h2/xxlarge945.gif" alt=" agr" align=" BASELINE" BORDER=" 0" > DPAL ECB Embryonic Chicken RCA-II Ricin Lectin |
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