Ganz P R, Pearlman R E
Eur J Biochem. 1980 Dec;113(1):159-73. doi: 10.1111/j.1432-1033.1980.tb06151.x.
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.
已从真核生物嗜热四膜虫中分离并鉴定出两种可能参与DNA复制的蛋白质。其中一种蛋白质，即DNA聚合酶，已被纯化至表观均一性。在有盐存在的情况下，该酶的天然分子量约为90000，在无盐时会聚集形成更高分子量的形式。当通过变性电泳分析该蛋白质时，纯化的酶制剂产生一个分子量为45000的主要亚基。嗜热四膜虫DNA聚合酶催化需要二价阳离子，与变性和天然DNA相比，更喜欢有缺口的模板引物。诸如聚（dT）·寡聚（A）这样的模板引物也可以被DNA聚合酶延长。然而，该酶不会将聚（A）·寡聚（dT）用作模板引物。巯基阻断试剂，如N-乙基马来酰亚胺，会抑制嗜热四膜虫DNA聚合酶。该DNA聚合酶缺乏可检测水平的单链和双链脱氧核糖核酸酶活性。在纯化的早期阶段，DNA聚合酶与一种分子量为100000的蛋白质一起进行色谱分离。当通过变性电泳分析时，这种蛋白质产生一个分子量为25000的单一主要多肽，具有单链DNA结合特性，并且能够在体外刺激DNA聚合酶催化的DNA合成的速率和程度。凭借后一种能力，该蛋白质被称为M（“修饰”）蛋白。使用含有大片段单链模板（如聚（dA）·（dT）10）且模板与引物比例为1:1混合的模板引物可实现对DNA聚合酶的最大刺激。M蛋白在体外对DNA聚合酶活性的刺激似乎涉及形成更长的产物DNA。
