Changes in DNA polymerases alpha, beta, and gamma during the replicative life span of cultured human fibroblasts.
Changes in DNA polymerases alpha, beta, and gamma during the replicative life span of cultured human fibroblasts.
复制标题
在培养的人成纤维细胞的复制寿命期间 DNA 聚合酶 α、β 和 γ 的变化。
DOI：
10.1021/bi00534a027
复制
发表时间：
1982
期刊：
影响因子：
2.9
通讯作者：
S. Linn
中科院分区：
文献类型：
--
作者：
S. W. Krauss;S. Linn
来自不同传代水平的 IMR-90 人二倍体成纤维细胞和 HeLa 细胞的 DNA 聚合酶被纯化并分级为 α 1、α 2、α 3、β 和 γ 物种和亚种，然后在 Mn2+ 或 Mg2+ 存在的情况下测量每个复制的合成模板引物的准确性。来自后期群体倍增水平的成纤维细胞的所有活动比来自早期群体倍增水平的相同聚合酶类型更频繁地掺入非互补三磷酸。 HeLa 聚合酶活性在 Mn2+ 存在下复制了几种不同的模板，其保真度高于群体倍增水平 27 或更高的成纤维细胞中的酶。从 IMR-90 细胞中提取的总 DNA 聚合酶活性随着群体倍增水平的增加而降低。 α-聚合酶活性通常随着群体倍增水平的增加而下降，而β-聚合酶活性保持相对恒定，除了在细胞复制寿命的最后阶段。此外，α2和α3的量相对于α1逐渐降低，并且在二乙氨基乙基纤维素色谱上出现了新的α型聚合酶活性α0。 HeLa 细胞还含有三种 α 物质，尽管其中两种从二乙氨基乙基纤维素中洗脱出来的磷酸盐浓度高于成纤维细胞中的 α 物质。相对于快速生长的细胞恢复的α聚合酶水平，汇合后群体倍增水平21的IMR-90细胞的α聚合酶水平降低。这种聚合酶活性具有一些类似于来自后期传代细胞的酶的色谱特性。此外，与从亚汇合细胞中分离的酶相比，来自这些细胞的 α、β 和 γ 聚合酶的保真度有所降低。
DNA polymerases from IMR-90 human diploid fibroblasts at various passage levels and from HeLa cells were purified and fractionated into alpha 1, alpha 2, alpha 3, beta, and gamma species and subspecies, and than the accuracy with which each one copied synthetic template-primers was measured in the presence of Mn2+ or Mg2+. All activities from fibroblasts of later population doubling levels incorporated noncomplementary triphosphates more frequently than did the same polymerase type from earlier population doubling levels. HeLa polymerase activities copied several different templates in the presence of Mn2+ with greater fidelity than enzymes from fibroblasts of population doubling level 27 or greater. The total DNA polymerase activity extracted from IMR-90 cells decreased with increasing population doubling levels. The alpha-polymerase activity generally declined with increasing population doubling levels, while beta-polymerase activity remained relatively constant, except at the very end of the cellular replicative life span. In addition, the amounts of alpha 2 and alpha 3 became progressively lower relative to alpha 1, and a new alpha-type polymerase activity, alpha 0, appeared upon diethylaminoethylcellulose chromatography. HeLa cells also contained three alpha species, though two of them eluted from diethylaminoethylcellulose at higher phosphate concentrations than alpha species from fibroblasts. Postconfluent IMR-90 cells of population doubling level 21 had a decreased level of alpha-polymerase relative to that recovered from rapidly growing cells. This polymerase activity had some chromatographic properties similar to enzyme from late-passage cells. In addition, the alpha-, beta-, and gamma-polymerases from these cells had decreased fidelities relative to those isolated from subconfluent cells.