Purification and general properties of the DNA-binding protein (P16) from rat liver mitochondria.
Purification and general properties of the DNA-binding protein (P16) from rat liver mitochondria.
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DOI：
10.1083/jcb.100.1.258
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发表时间：
1985-01
期刊：
影响因子：
--
通讯作者：
Van Tuyle GC
中科院分区：
文献类型：
--
作者：
Pavco PA;Van Tuyle GC
采用单链DNA琼脂糖亲和层析法从大鼠肝脏线粒体裂解物中分离出线粒体DNA结合蛋白P16，并在制备过程中采用碱性CsCl等重梯度法分离出线粒体DNA。根据SDS PAGE，梯度的顶部部分含有单个多肽物种（Mr大约等于15,200）。用蛋白酶K消化单链dna结合的P16产生蛋白酶不敏感的dna结合片段（Mr大约等于6000），该片段的纯化过程与完整的P16基本相同。采用常规方法获得P16和dna结合片段的部分氨基酸组成。亚细胞部分的分析显示，几乎所有的细胞P16都位于线粒体中，只有微量的电泳迁移率相当的蛋白质可以从细胞核或细胞质部分分离出来。在原代培养的大鼠肝细胞中，用[35S]甲硫氨酸标记P16被细胞质翻译抑制剂环己亚胺抑制了90%以上，但不受线粒体特异性药物氯霉素的影响。这些结果表明P16是在细胞质核糖体上合成并进入线粒体的。将纯化的P16添加到去蛋白化的线粒体DNA中，可以完全保护标记的移位环的新生链，防止在SstI切割亲本DNA时发生分支迁移损失，从而提供了强有力的证据，证明P16是体外功能所需的单一实体。P16与单链phi X174 DNA、双链（RF） phi X174 DNA或大肠杆菌核糖体RNA孵育，随后对结合蛋白的核酸种类进行分析表明，P16对单链DNA有强烈的偏好，对RNA或双链DNA没有可检测到的亲和力。通过直接电子显微镜检查p16 -单链phi X174 DNA复合物，发现蛋白质相关单链DNA特征的增厚，不规则纤维。
The mitochondrial DNA-binding protein P16 was isolated from rat liver mitochondrial lysates by affinity chromatography on single strand DNA agarose and separated from DNA in the preparation by alkaline CsCl isopycnic gradients. The top fraction of the gradients contained a single polypeptide species (Mr approximately equal to 15,200) based upon SDS PAGE. Digestion of single strand DNA-bound P16 with proteinase K produced a protease-insensitive, DNA-binding fragment (Mr approximately equal to 6,000) that has been purified by essentially the same procedures used for intact P16. The partial amino acid compositions for P16 and the DNA-binding fragment were obtained by conventional methods. Analysis of subcellular fractions revealed that nearly all of the cellular P16 was located in the mitochondria and that only trace amounts of protein of comparable electrophoretic mobility could be isolated from the nuclear or cytoplasmic fractions. The labeling of P16 with [35S]methionine in primary rat hepatocyte cultures was inhibited by more than 90% by the cytoplasmic translation inhibitor cycloheximide, but unaffected by the mitochondrial-specific agent chloramphenicol. These results indicate that P16 is synthesized on cytoplasmic ribosomes and imported into the mitochondria. The addition of purified P16 to deproteinized mitochondrial DNA resulted in the complete protection of the labeled nascent strands of displacement loops against branch migrational loss during cleavage of parental DNA with SstI, thus providing strong evidence that P16 is the single entity required for this in vitro function. Incubation of P16 with single strand phi X174 DNA, double strand (RF) phi X174 DNA, or Escherichia coli ribosomal RNA and subsequent analysis of the nucleic acid species for bound protein indicated a strong preference of P16 for single strand DNA and no detectable affinity for RNA or double strand DNA. Examination of P16-single strand phi X174 DNA complexes by direct electron microscopy revealed thickened, irregular fibers characteristic of protein-associated single strand DNA.