Purification of a DNA polymerase-DNA primase complex from calf thymus glands.
An immunoabsorbent column, prepared by covalently linking mouse monoclonal anti-calf thymus DNA polymerase-alpha to Protein A-Sepharose, was used as the primary purification step for rapid isolation of DNA polymerase-alpha from calf thymus-gland extracts. In a 4-step procedure consisting of the removal of nucleic acids by protamine sulfate precipitation, chromatography on the immunoabsorbent column, desalting on Sephadex G-50, and removal of bovine immunoglobulins on Protein A-Sepharose, DNA polymerase-alpha activity was purified about 5000-fold from the crude extract with greater than 40% recovery of total enzyme activity. The antibody column-purified DNA polymerase-alpha fraction contains a DNA primase activity that is efficient in replication of single-stranded DNA and poly(dT) when rNTPs are included in the replication reactions. Synthesis by calf thymus DNA polymerase-primase is totally dependent on added template. Complete replication of circular single-stranded phage DNA is achieved with polymerase-primase producing a nicked circular DNA containing oligoribonucleotide primer in the final product. Primers synthesized with single-stranded phage DNA as template were up to 10 nucleotides long when dNTPs were omitted from the reaction and 8 or less nucleotides long when dNTPs were present. Primers synthesized using poly(dT) consisted of three populations when dATP was absent from the reaction, averaging 20 nucleotides, 10 nucleotides, and 3-4 nucleotides. The 20-nucleotide population was not found when dATP was included in the reaction.
D000818:Animals; D002417:Cattle; D004257:DNA Polymerase II / Q000302:isolation & purification* / Q000378:metabolism; D019915:DNA Primase; D004261:DNA Replication; D004262:DNA Restriction Enzymes; D007700:Kinetics; D008970:Molecular Weight; D009097:Multienzyme Complexes / Q000302:isolation & purification* / Q000378:metabolism; D012316:RNA Nucleotidyltransferases / Q000302:isolation & purification*; D013698:Templates, Genetic; D013950:Thymus Gland / Q000201:enzymology*
D009097:Multienzyme Complexes; D019915:DNA Primase; D012316:RNA Nucleotidyltransferases; D004257:DNA Polymerase II; D004262:DNA Restriction Enzymes