DNA polymerase-associated lectin (DPAL) and its binding to the galactose-containing glycoconjugate of the replication complex.
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.
Kelley
T J
TJ
Department of Pediatrics, CaseWestern Reserve University, Cleveland, OH 44106, USA.
St Amand
T
T
Groll
J M
JM
Ray
S
S
Basu
S
S
D000818:Animals; D020134:Catalytic Domain; D002642:Chick Embryo; D002851:Chromatography, High Pressure Liquid; D003208:Concanavalin A / Q000737:chemistry; D004256:DNA Polymerase I / Q000737:chemistry*; D019915:DNA Primase / Q000737:chemistry; D004261:DNA Replication; D005690:Galactose / Q000737:chemistry*; D006001:Glycoconjugates / Q000737:chemistry*; D006801:Humans; D037102:Lectins / Q000737:chemistry*; D008756:Methylgalactosides / Q000737:chemistry; D008970:Molecular Weight; D011233:Precipitin Tests; D011485:Protein Binding; D051381:Rats; D014118:Toxins, Biological / Q000737:chemistry; D014407:Tumor Cells, Cultured
D006001:Glycoconjugates; D037102:Lectins; D008756:Methylgalactosides; C024930:RCA II; D014118:Toxins, Biological; D003208:Concanavalin A; C031552:methyl beta-galactoside; D019915:DNA Primase; D004256:DNA Polymerase I; D005690:Galactose