Purification from Tetrahymena thermophila of DNA polymerase and a protein which modifies its activity.
Two proteins, which may be involved in DNA replication, have been isolated and characterized from the eukaryote Tetrahymena thermophila. One of these proteins, DNA polymerase, has been purified to apparent homogeneity. The enzyme has a native molecular weight of approximately 90 000 in the presence of salt and aggregates to higher-molecular-weight forms in the absence of salt. Purified preparations of the enzyme yield a major subunit of Mr 45 000 when the protein is analyzed by denaturing electrophoresis. Tetrahymena DNA polymerase requires a divalent cation for catalysis and prefers gapped template-primers over denatured and native DNAs. A template-primer such as poly(dT) . oligo(A) can also be elongated by the DNA polymerase. However, the enzyme will not use poly(A) . oligo(dT) as a template-primer. Sulfhydryl-blocking reagents, such as N-ethylmaleimide, inhibit Tetrahymena DNA polymerase. The DNA polymerase lacks assayable levels of both single and double-stranded deoxyribonuclease activity. Throughout the early stages of purification the DNA polymerase chromatographs together with a protein of molecular weight 100 000. This protein, which yields a single major polypeptide of Mr 25 000 when analyzed by denaturing electrophoresis, has single-stranded-DNA-binding properties and has the ability to stimulate both the rate and extent of DNA-polymerase-catalyzed DNA synthesis in vitro. By virtue of this latter ability, the protein has been referred to as the M (for 'modifying') protein. Maximum stimulation of DNA polymerase was achieved with template-primers, which contained large stretches of single-stranded template such as poly(dA) . (dT)10 mixed in a template-to-primer ratio of one to one. Stimulation of DNA polymerase activity by M protein in vitro appears to involve formation of longer product DNA.
D002352:Carrier Proteins / Q000302:isolation & purification* / Q000378:metabolism; D002499:Centrifugation, Density Gradient; D002848:Chromatography, DEAE-Cellulose; D002850:Chromatography, Gel; D004259:DNA-Directed DNA Polymerase / Q000302:isolation & purification* / Q000378:metabolism; D004591:Electrophoresis, Polyacrylamide Gel; D008970:Molecular Weight; D011067:Poly dA-dT / Q000378:metabolism; D015800:Protozoan Proteins; D013379:Substrate Specificity; D013698:Templates, Genetic; D013768:Tetrahymena / Q000201:enzymology*
D002352:Carrier Proteins; C028598:DNA polymerase-modifying protein, Tetrahymena thermophila; D015800:Protozoan Proteins; D011067:Poly dA-dT; D004259:DNA-Directed DNA Polymerase