Biomaterial Database

Purification of a DNA polymerase-DNA primase complex from calf thymus glands. 1984

L M Chang, and E Rafter, and C Augl, and F J Bollum

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.

UI	MeSH Term	Description	Entries
D007700	Kinetics	The rate dynamics in chemical or physical systems.
D008970	Molecular Weight	The sum of the weight of all the atoms in a molecule.	Molecular Weights,Weight, Molecular,Weights, Molecular
D009097	Multienzyme Complexes	Systems of enzymes which function sequentially by catalyzing consecutive reactions linked by common metabolic intermediates. They may involve simply a transfer of water molecules or hydrogen atoms and may be associated with large supramolecular structures such as MITOCHONDRIA or RIBOSOMES.	Complexes, Multienzyme
D002417	Cattle	Domesticated bovine animals of the genus Bos, usually kept on a farm or ranch and used for the production of meat or dairy products or for heavy labor.	Beef Cow,Bos grunniens,Bos indicus,Bos indicus Cattle,Bos taurus,Cow,Cow, Domestic,Dairy Cow,Holstein Cow,Indicine Cattle,Taurine Cattle,Taurus Cattle,Yak,Zebu,Beef Cows,Bos indicus Cattles,Cattle, Bos indicus,Cattle, Indicine,Cattle, Taurine,Cattle, Taurus,Cattles, Bos indicus,Cattles, Indicine,Cattles, Taurine,Cattles, Taurus,Cow, Beef,Cow, Dairy,Cow, Holstein,Cows,Dairy Cows,Domestic Cow,Domestic Cows,Indicine Cattles,Taurine Cattles,Taurus Cattles,Yaks,Zebus
D004257	DNA Polymerase II	A DNA-dependent DNA polymerase characterized in E. coli and other lower organisms. It may be present in higher organisms and has an intrinsic molecular activity only 5% of that of DNA Polymerase I. This polymerase has 3'-5' exonuclease activity, is effective only on duplex DNA with gaps or single-strand ends of less than 100 nucleotides as template, and is inhibited by sulfhydryl reagents.	DNA Polymerase epsilon,DNA-Dependent DNA Polymerase II,DNA Pol II,DNA Dependent DNA Polymerase II
D004261	DNA Replication	The process by which a DNA molecule is duplicated.	Autonomous Replication,Replication, Autonomous,Autonomous Replications,DNA Replications,Replication, DNA,Replications, Autonomous,Replications, DNA
D004262	DNA Restriction Enzymes	Enzymes that are part of the restriction-modification systems. They catalyze the endonucleolytic cleavage of DNA sequences which lack the species-specific methylation pattern in the host cell's DNA. Cleavage yields random or specific double-stranded fragments with terminal 5'-phosphates. The function of restriction enzymes is to destroy any foreign DNA that invades the host cell. Most have been studied in bacterial systems, but a few have been found in eukaryotic organisms. They are also used as tools for the systematic dissection and mapping of chromosomes, in the determination of base sequences of DNAs, and have made it possible to splice and recombine genes from one organism into the genome of another. EC 3.21.1.	Restriction Endonucleases,DNA Restriction Enzyme,Restriction Endonuclease,Endonuclease, Restriction,Endonucleases, Restriction,Enzymes, DNA Restriction,Restriction Enzyme, DNA,Restriction Enzymes, DNA
D000818	Animals	Unicellular or multicellular, heterotrophic organisms, that have sensation and the power of voluntary movement. Under the older five kingdom paradigm, Animalia was one of the kingdoms. Under the modern three domain model, Animalia represents one of the many groups in the domain EUKARYOTA.	Animal,Metazoa,Animalia
D012316	RNA Nucleotidyltransferases	Enzymes that catalyze the template-directed incorporation of ribonucleotides into an RNA chain. EC 2.7.7.-.	Nucleotidyltransferases, RNA
D013698	Templates, Genetic	Macromolecular molds for the synthesis of complementary macromolecules, as in DNA REPLICATION; GENETIC TRANSCRIPTION of DNA to RNA, and GENETIC TRANSLATION of RNA into POLYPEPTIDES.	Genetic Template,Genetic Templates,Template, Genetic