Establishment of the basis for developing inhibitors that target the DNA helicase involved in DNA replication

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The aim of this project is to provide the basis for developing new anti-cancer drugs by identifying DNA helicase involved in DNA replication in mammalian cells and by developing screening systems for inhibitors of the helicase. We have succeeded to identify the DNA helicase involved in DNA replication in mammalian cells by obtaining following results.1. In tsFT848 cells that are temperature-sensitive mutants for DNA replication, the decrease in the level of DNA synthesis correlated with the decrease in the level of DNA-dependent ATP ase activity of DNA helicase B.2. DNA helicase B from tsFT848 cells was more heat-sensitive than that from wild-type cells.3. One Missense mutation from Phe to Cys occurred in the DNA helicase B of mutant cells at the amino acid position, 724.4.DNA helicase B stimulated DNA primase activity.5.DNA helicase B functioned as a helicase in a cell-free DNA replication system.6. DNA helicase B was localized at the replication foci.As to the development of screening systems, we developed a system measuring DNA-dependent ATPase activity instead of DNA helicase activity with a platereader, which was able to deal with many samples at once. To supply the target helicase to the screening system, we have tried to express DNA helicase B in Escherichia coli and yeast but not succeeded yet. We are now trying to obtain large amount of DNA helicase B by using the baculovirus system.Although we have not succeeded to find inhibitors for DNA helicase B,we think that we could provide the basis for developing new anti-cancer drugs by identifying the target and developing the screening system.