Identification and purification of a factor that binds to the Mlu I cell cycle box of yeast DNA replication genes.

In Saccharomyces cerevisiae, the genes encoding at least 10 enzymes involved in DNA replication are periodically expressed in the late G1 and S phases of the cell cycle. All of these genes have one copy or more of the sequence ACGCGT, which conforms to the recognition site for the Mlu I restriction endonuclease. For the CDC21, CDC9, and POL1 genes, the Mlu I site has been shown to be absolutely required for periodic transcription. Using nuclear extracts fractionated by conventional and oligonucleotide affinity chromatography, we have purified a 17-kDa protein that recognizes the Mlu I motif. Synthetic oligonucleotides containing mutated Mlu I sites do not bind the protein. In contrast, synthetic oligonucleotides derived from the CDC2, CDC6, and CDC21 genes, which are expressed with the same timing as POL1, bind purified protein efficiently. IMAGES:

在酿酒酵母（Saccharomyces cerevisiae）中，至少10种参与DNA复制的酶的编码基因会在细胞周期的晚G1期与S期周期性表达。所有这类基因均含有一个或多个ACGCGT序列，该序列恰好是限制性内切酶Mlu I的识别位点。针对CDC21、CDC9与POL1基因，已有研究证实Mlu I位点是其周期性转录所绝对必需的元件。本研究利用经常规层析与寡核苷酸亲和层析分级分离的细胞核提取物，纯化得到一种可识别Mlu I基序的17 kDa蛋白质。含有突变型Mlu I位点的合成寡核苷酸无法与该蛋白质结合。与之相反，从CDC2、CDC6与CDC21基因（其表达时序与POL1一致）衍生而来的合成寡核苷酸，可高效结合纯化得到的该蛋白质。图像：