DNA Polymerase-Associated Lectin (DPAL) and Its Binding to the Galactose-Containing Glycoconjugate of the Replication Complex

摘要

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 form IMPR-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 form a single primary structure. Immunoblot analysis whit 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 and #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 120kDa in 19-day-old ECB [Ray, aS.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 (M_r = 186kDa) 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.