Establishing the human rolling circle reaction

In eukaryotes, the complex comprised of Mcm2–7, Cdc45 and GINS (CMG) is essential for DNA replication. Several lines of evidence indicate that the Mcm2–7 complex is the motor of the replicative helicase (reviewed in ref. 1), which is activated by its association with Cdc45 and GINS. Recently, we described the isolation and characterization of the human (h) CMG complex. In HeLa cells, this complex was formed only on chromatin and, following its isolation from cells, exhibited DNA helicase activity. Purified from Sf9 cells, hCMG possesses 3'→5' DNA helicase activity, indicating that it moves ahead of the leading-strand DNA polymerase (pol). In contrast, the prokaryotic helicase DnaB, which unwinds DNA in the 5'→3' direction, moves on the lagging strand. Detailed information about the progression of the prokaryotic replication fork was obtained using the rolling-circle method (ref. 4 and references therein). These studies permitted a detailed characterization of the joint action of the replicative pol and replicative helicase. In the rolling-circle reaction, the pol extends the 3' end of a primer annealed to a minicircle that is then unwound simultaneously by the helicase (for a possible arrangement of proteins at the replication fork, see Fig. 1). The emerging single-stranded 5'-tail provides the template for lagging-strand synthesis. In most experiments, minicircles were engineered to contain only three nucleotides, allowing the distinction between leadingand lagging-strand nucleotide incorporation. We initiated experiments to develop a eukaryotic replication fork in order to Establishing the human rolling circle reaction