Concerted mechanism of DNA replication initiation and transcription in mammalian cells

虽然利用AIMMA病毒系统已经研究了DNA复制和转录的协同机制，但在细胞系统中的协同机制仍不清楚。我们使用c-myc、N-myc、免疫球蛋白重链(IgH)、热休克蛋白70(HSP70)、P53、重复序列Alu和SV40等系统来解决这种机制。我们已经确定了活细胞中DNA复制(ORI)功能的起源。C-myc基因转录起始点上游15~20kb，HSP70基因启动子，IgH基因增强，p53基因第11外显子下游10kb。含有上述区域的克隆可以在哺乳动物细胞中自主复制(ARS)，并且它们的区域与转录调控区域重叠。因此，这些结果清楚地表明了哺乳动物细胞中DNA复制启动和转录的协同机制。然后，我们鉴定了识别上述区域的蛋白质因子。MSSP识别c-myc基因的ORI/增强子，HSBP识别HSP70基因的启动子/ORI，两者都与c-myc蛋白形成复合体。克隆了MSSP的两个cDNA。Oct-1还与IgH基因中的增强子/ori结合。在N-myc基因中，45K和110K蛋白结合到作用ARS的增强子区域。我们还鉴定了与SV40的ORI核心中的AT富含序列结合的蛋白质Soap，并且Soap与MSSP-1很可能是相同的。Alu家族DNA调控DNA复制和转录，并被37K蛋白识别。这里描述的所有蛋白质都与序列结合--特异性地与双链和单链DNA结合，并可能参与DNA复制和转录。

Although the concerted mechanism of DNA replivation and transcription has been studied by using anima virus system, that in cellular system is stil not clarified. We have used the systems including c-myc, N-myc, immunogloburin heavy chain (IgH), heat shock protein 70 (hsp70), P53, repeated sequence Alu, and SV40 to dissolve such mechanisms.We have determined the origin of DNA replication (ori) functioning in living cells. The origins identified were located as following; 15-20 kbs upstream of the start site of transcription in c-myc gene, promoter in hsp70 gene, enhance in IgH gene, 10 kb downstream of the 11exon in p53 gene. Clones containing above regions could replicate autonomously (ARS) in mammalian cells and their regions overlapped the transcriptional regulatory regions. Thus these results clearly indicate the concerted mechanism of DNA replication initiation and transcription in mammalian cells.We then identified the protein factors recognizing the regions described above. MSSP recognized the ori/enhancer in c-myc gene and HSBP did the promoter/ori in hsp70 gene, both of which made complexes with c-myc protein. Two cDNAs of MSSP were cloned. Oct-1 also bound to the enhancer/ori in IgH gene. In N-myc gene, 45k and 110 K proteins bound to the enhancer region acting ARS. We have also identified the protein, SOAP, which bound AT rich sequence placed in ori core of SV40, and SOAP and MSSP-1 are most probably identical each other. Alu family DNA modulated both DNA replication and transcription, and was recognized by 37 K protein. All the proteins described here bind sequenece-specifically to both double and single stranded DNA and may be involved in DNA replication and transcription.