Induction of DNA replication by transcription in the region upstream of the human c-myc gene in a model replication system.
An important relationship between transcription and initiation of DNA replication in both eukaryotes and prokaryotes has been suggested. In an attempt to understand the molecular mechanism of this interaction, we examined whether transcription can induce DNA replication in vitro by constructing a system in which both replication and transcription were combined. Relaxed circular DNA possessing a replication initiation zone located upstream of the human c-myc gene and a T7 promoter near the P1 promoter of the gene was replicated in the presence of T7 RNA polymerase. In our model system, replication was carried out with the proteins required for simian virus 40 DNA replication. DNA synthesis, which was dependent on both T7 RNA polymerase and the replication proteins, was detected mainly in the promoter and upstream regions of the c-myc gene. Blocking RNA synthesis at the initial stage of the reaction severely reduced DNA synthesis, suggesting that RNA chain elongation is required to induce DNA synthesis. The results indicated that transcription can induce DNA replication in the upstream region of the transcribed gene, most likely by introducing negative supercoiling into the region, which results in unwinding of the DNA duplex.
Ohba
R
R
Mitsubishi Kasei Institute of Life Sciences, Tokyo, Japan.
Matsumoto
K
K
Ishimi
Y
Y
D017123:Bacteriophage T7 / Q000235:genetics; D015342:DNA Probes; D004261:DNA Replication; D004270:DNA, Circular / Q000096:biosynthesis; D004278:DNA, Superhelical / Q000378:metabolism; D012321:DNA-Directed RNA Polymerases / Q000378:metabolism; D016259:Genes, myc; D006801:Humans; D008957:Models, Genetic; D010957:Plasmids; D011401:Promoter Regions, Genetic; D012045:Regulatory Sequences, Nucleic Acid; D015183:Restriction Mapping; D017384:Sequence Deletion; D013539:Simian virus 40 / Q000235:genetics; D014158:Transcription, Genetic; D014764:Viral Proteins
D015342:DNA Probes; D004270:DNA, Circular; D004278:DNA, Superhelical; D014764:Viral Proteins; C062210:bacteriophage T7 RNA polymerase; D012321:DNA-Directed RNA Polymerases