The crucial job of ensuring that just one copy of a genome gets made during cell division turns out to be shared by two independent "controllers," researchers from the School of Medicine report in the Oct. 23 issue of the Proceedings of the National Academy of Sciences.

In experiments with yeast cells, the scientists discovered that if the two controller proteins remain in the cell, then copying continues abnormally. Normally, the proteins are destroyed after a single copy, or replication, of the DNA is made.

"We knew these proteins were required for DNA replication and that they normally went away after one DNA copy was made, but we didn't know whether their disappearance was important for controlling the duplication of the genome," says Thomas Kelly, the Boury Professor of Molecular Biology and Genetics and director of the Institute for Basic Biomedical Sciences. "Now we know that DNA replication ceases because these two proteins are destroyed."

Understanding this complex and tightly regulated process may help clarify what goes wrong in cancer cells, say the researchers. The yeast used in the experiment (Schizosaccharomyces pombe) divides by splitting into two new cells, each with a copy of the organism's entire genome, in a process very similar to that of human cells.

"In human cells, for reasons still largely unknown, some cells ultimately acquire enough genetic mutations to cause cancer, despite having different ways to prevent, find and fix problems in the genome," says Mark Frattini, a postdoctoral fellow in the School of Medicine's Department of Medical Oncology and Department of Molecular Biology and Genetics, which is part of the school's Institute for Basic Biomedical Sciences. "We can't yet link these two controller proteins in yeast or their human counterparts to cancer, but we do have a new genome control pathway to examine."

In the yeast, one controller, the Cdc18 protein, helps build machinery that copies DNA, and the other, Cdt1, helps start that machine. The levels of both proteins normally rise before DNA replication and fall once it's completed.

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," Frattini says.

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, Frattini says. 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.

In addition to Kelly and Frattini, co-authors are first author Vidya Gopalakrishnan, Pamela Simancek and Christopher Houchens, all of the School of Medicine; Hilary Snaith, formerly of the Salk Institute and now at the University of Edinburgh; and Shelley Sazer, of the Baylor College of Medicine.