By disrupting the yeasts' ability to regulate the levels of Cdc18 and Cdt1, the Hopkins scientists proved that the normal destruction of the two proteins restricts DNA replication to a single copy. In cells with mutant Cdc18 and Cdt1 whose levels never drop, DNA replication keeps going.
"Having two proteins offers redundant protection against making extra copies of DNA, which helps maintain the integrity of the genome for subsequent generations of cells," says Frattini.
The scientists used a version of the gene for Cdc18 that would produce normal amounts of an altered protein that could not be marked for destruction, but which would otherwise function normally. In the case of Cdt1, the scientists created two new versions of the gene, both of which were controlled by an "on switch" that the researchers, but not the cell, could manipulate.
The mutations worked as expected: the levels of Cdc18 and the Cdt1 proteins no longer varied normally. Cdc18 levels increased when the cell began constructing the DNA copier, as normal, but then remained high. And all cells produced Cdt1, regardless of their point in cell division.
The amount of DNA in the cells reflected the changes' effects on the DNA copier. Many cells with the mutant Cdc18 and Cdt1 contained as much as two to four times the amount of DNA they should have, says Frattini. Cells with just one of the proteins altered were normal.
Other researchers have found that very large amounts of indestructible Cdc18 could lead to extra replication of DNA in the yeast, but Hopkins' experiments show that if protein levels are closer to normal, both Cdc18 and Cdt1 are required to continue DNA copying.