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Properties of the human Cdc45/Mcm2-7/GINS helicase complex and its action with DNA polymerase Îµ in rolling circle DNA synthesis
- Kang, Young-Hoon, Galal, Wiebke Chemnitz, Farina, Andrea, Tappin, Inger, Hurwitz, Jerard
- Proceedings of the National Academy of Sciences of the United States of America 2012 v.109 no.16 pp. 6042-6047
- DNA helicases, DNA-directed DNA polymerase, Escherichia coli, adenosine triphosphate, binding proteins, circular DNA, humans, magnesium, single-stranded DNA
- In eukaryotes, although the Mcm2-7 complex is a key component of the replicative DNA helicase, its association with Cdc45 and GINS (the CMG complex) is required for the activation of the DNA helicase. Here, we show that the CMG complex is localized to chromatin in human cells and describe the biochemical properties of the human CMG complex purified from baculovirus-infected Sf9 cells. The isolated complex binds to ssDNA regions in the presence of magnesium and ATP (or a nonhydrolyzable ATP analog), contains maximal DNA helicase in the presence of forked DNA structures, and translocates along the leading strand (3' to 5' direction). The complex hydrolyses ATP in the absence of DNA; unwinds duplex regions up to 500 bp; and either replication protein A or Escherichia coli single stranded binding protein increases the efficiency of displacement of long duplex regions. Using a 200-nt primed circular DNA substrate, the combined action of human DNA polymerase Îµ and the human CMG complex leads to the formation of products >10 kb in length. These findings suggest that the coordinated action of these replication complexes supports leading strand synthesis.