Concerted mechanism of DNA replication initiation and transcription in mammalian cells

虽然利用动物病毒系统研究了DNA复制与转录的协同机制，但细胞系统中的机制尚不清楚。我们用c-myc、N-myc、免疫球蛋白重链（IgH）、热休克蛋白70（hsp 70）、P53、重复序列Alu和SV 40等系统来解释这些机制，并确定了活细胞中DNA复制的起始点（ori）。经鉴定，其起始位点位于c-myc基因转录起始位点上游15-20 kbs，启动子位于hsp 70基因，增强子位于IgH基因，p53基因第11外显子下游10 kb。含有上述区域的克隆能在哺乳动物细胞中自主复制（ARS），其区域与转录调控区重叠。这些结果清楚地表明了哺乳动物细胞中DNA复制起始和转录的协同机制。MSSP识别c-myc基因的ori/enhancer，HSBP识别hsp 70基因的promoter/ori，两者均与c-myc蛋白形成复合物。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，45 K和110 K蛋白与ARS的增强子区域结合。我们还鉴定了与SV 40 ori核心AT丰富序列结合的蛋白SOAP，SOAP和MSSP-1很可能是同一种蛋白。Alu家族DNA对DNA的复制和转录均有调节作用，并被37 K蛋白识别.本文所述的所有蛋白质均与双链和单链DNA序列特异性结合，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.