DNA replication from initiation zones of mammalian cells in a model system

Abstract

We reported that DNA replication initiates from the region containing an autonomously replicating sequence from Saccharomyces cerevisiae when negatively supercoiled plasmid DNA is incubated with the proteins required for simian virus 40 DNA replication (Y. Ishimi and K. Matsumoto, Proc. Natl. Acad. Sci. USA 90:5399-5403, 1993). In this study, the DNAs containing initiation zones from mammalian cells were replicated in this model system. When negatively supercoiled DNA containing an initiation zone (2 kb) upstream of the human c-myc gene was incubated with simian virus 40 T antigen as a DNA helicase, HSSB (also called replication protein A), and DNA polymerase alpha-primase complex isolated from HeLa cells, DNA replication was specifically initiated from the center of the initiation zone, which was elongated bidirectionally in the presence of a DNA swivelase. Without HSSB, the level of DNA synthesis was significantly reduced and the localized initiation could not be detected, indicating that HSSB plays an essential role in the initiation of DNA replication. The digestion of negatively supercoiled template DNA with a single-strand-specific nuclease revealed that HSSB stimulated DNA unwinding in the center of the initiation zone where the DNA duplex is relatively unstable. In contrast, DNA replication started from a broad region of an initiation zone downstream of the dihydrofolate reductase gene from chinese hamster ovary cells, but the center of the region was mapped near the origin of bidirectional DNA replication. These results suggested that this system mimics a fundamental process of initiation of eukaryotic DNA replication. The mechanism of initiation is discussed.