Kuno K, Murakami S, Kuno S
Department of Biochemistry, School of Medicine, Kanazawa University, Ishikawa, Japan.
Gene. 1990 Oct 30;95(1):73-7. doi: 10.1016/0378-1119(90)90415-n.
Since plasmids containing autonomously replicating sequence(s) (ARS) can transform Saccharomyces cerevisiae cells at high frequency, ARS are considered to be the replication origins of chromosomes. To study the mechanism of initiation of eukaryotic chromosomal replication, we examined protein factors which interact with the ARS1 region located near the centromere of chromosome IV in S. cerevisiae. Using the gel-shift assay, we found protein factors which bound to a single-stranded, 97-bp fragment of the ARS1 region containing the core consensus. Competition experiments with various oligodeoxyribonucleotides (oligos) suggest that a site recognized by the factor(s) was within the element containing the core consensus and adjacent close matches to the core consensus of the minus strand. Indeed, when the oligo containing the minus strand of this element was used as a probe, two oligo-protein complexes were detected. Mutations in the core consensus reduced these binding activities. When the plus-strand oligo of the same region was used as a probe, a retarded band was also detected, but with less specific binding. Considering the fact that the core consensus and close matches to the core consensus are important for ARS function, these results imply that the protein factors detected in this experiment may participate in DNA replication.
由于含有自主复制序列（ARS）的质粒能够高频转化酿酒酵母细胞，因此ARS被认为是染色体的复制起点。为了研究真核染色体复制的起始机制，我们检测了与酿酒酵母中位于第四条染色体着丝粒附近的ARS1区域相互作用的蛋白质因子。使用凝胶迁移实验，我们发现了与包含核心共有序列的ARS1区域的单链97碱基对片段结合的蛋白质因子。用各种寡脱氧核糖核苷酸（oligos）进行的竞争实验表明，该因子识别的位点位于包含核心共有序列以及与负链核心共有序列相邻紧密匹配的元件内。实际上，当使用包含该元件负链的oligo作为探针时，检测到了两种oligo-蛋白质复合物。核心共有序列中的突变降低了这些结合活性。当使用同一区域的正链oligo作为探针时，也检测到了一条滞后带，但特异性结合较少。考虑到核心共有序列以及与核心共有序列的紧密匹配对ARS功能很重要，这些结果表明在本实验中检测到的蛋白质因子可能参与DNA复制。
