Hirose F, Yamaguchi M, Handa H, Inomata Y, Matsukage A
Laboratory of Cell Biology, Aichi Cancer Center Research Institute, Nagoya, Japan.
J Biol Chem. 1993 Jan 25;268(3):2092-9.
Upstream regions containing a novel common 8-base pair (bp) palindromic sequence, 5'-TATCGATA (Drosophila DNA replication-related element (DRE)), are required for the high expression of Drosophila genes for DNA polymerase alpha and the proliferating cell nuclear antigen (PCNA) (an auxiliary protein for DNA polymerase delta). Three DREs and one DRE are present in the DNA polymerase alpha gene (nucleotides-217, -83, and -30 with respect to the transcription initiation site) and in the PCNA gene (nucleotide-100), respectively. Deletions or 2-bp insertional mutations of DRE sequences led to an extensive reduction of promoter activities of both genes. Chemically synthesized oligonucleotides containing DRE sequences greatly stimulated the activity of the heterologous promoter of the Drosophila metallothionein gene, in addition to the promoter of the PCNA gene, when they were placed upstream from these promoters in a normal or a reverse orientation. The stimulatory effect increased synergistically and depended on the number of DREs. DRE activated the promoter when placed within 1.4 kilobases upstream from the promoter, but was much less active when placed 2.5 kilobases or more apart from the promoter. Using a gel mobility shift assay method, we obtained evidence for a protein factor (DREF) in the nuclear extract of cultured Drosophila cells (Kc cells), and this factor specifically binds to DREs of both genes. DNase I footprinting analysis indicated that DREF binds to the 24-bp DRE region of the DNA polymerase alpha gene in which 8-bp palindromic sequences are centered. A UV cross-linking experiment revealed that a polypeptide of approximately 90 kDa in the nuclear extract interacts directly with the DRE sequence. Using DRE-conjugated latex particles, DREF was affinity-purified from the Kc cell nuclear extract. By comparing results obtained by SDS-polyacrylamide gel electrophoresis and gel mobility shift experiments, we concluded that DREF is associated with the 86-kDa polypeptide. On gel filtration chromatography, a single peak of DREF activity was recovered in fractions corresponding to a molecular mass of 170 kDa, and the 86-kDa polypeptide was detected only in the corresponding fractions; thus, active DREF is probably a homodimeric form of the 86-kDa polypeptide. DREF may play important roles in coordinating expressions of Drosophila DNA replication-related genes.
包含一个新的8碱基对（bp）回文序列5'-TATCGATA（果蝇DNA复制相关元件（DRE））的上游区域，对于果蝇DNA聚合酶α基因和增殖细胞核抗原（PCNA）（DNA聚合酶δ的辅助蛋白）基因的高表达是必需的。在DNA聚合酶α基因（相对于转录起始位点的核苷酸-217、-83和-30）和PCNA基因（核苷酸-100）中分别存在三个DRE和一个DRE。DRE序列的缺失或2碱基插入突变导致两个基因的启动子活性大幅降低。当化学合成的含DRE序列的寡核苷酸以正常或反向方向置于果蝇金属硫蛋白基因的异源启动子以及PCNA基因的启动子上游时，它们极大地刺激了这些启动子的活性。刺激作用协同增加且取决于DRE的数量。DRE置于启动子上游1.4千碱基内时可激活启动子，但置于与启动子相距2.5千碱基或更远时活性则低得多。使用凝胶迁移率变动分析方法，我们在培养的果蝇细胞（Kc细胞）的核提取物中获得了一种蛋白因子（DREF）的证据，该因子特异性结合两个基因的DRE。DNA酶I足迹分析表明DREF结合到DNA聚合酶α基因的24 bp DRE区域，其中8 bp回文序列位于中心。紫外线交联实验显示核提取物中一种约90 kDa的多肽直接与DRE序列相互作用。使用DRE偶联的乳胶颗粒，从Kc细胞核提取物中亲和纯化出DREF。通过比较SDS-聚丙烯酰胺凝胶电泳和凝胶迁移率变动实验得到的结果，我们得出结论DREF与86 kDa多肽相关。在凝胶过滤色谱上，在对应于分子量170 kDa的级分中回收了单一峰的DREF活性，并且仅在相应级分中检测到86 kDa多肽；因此，活性DREF可能是86 kDa多肽的同源二聚体形式。DREF可能在协调果蝇DNA复制相关基因的表达中发挥重要作用。
