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 90000 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 M r 45000 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 1. This protein, which yields a single major polypeptide of M r 25000 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.