October 22, 2001
MEDIA CONTACT: Joanna Downer
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 Johns Hopkins 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
"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, M.D., Ph.D., professor and director 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
"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, M.D.,
Ph.D., a postdoctoral fellow in the department of medical oncology and the 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," 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.
In addition to Kelly and Frattini, co-authors are first author Vidya Gopalakrishnan, Pamela Simancek and Christopher Houchens of The Johns Hopkins University School of Medicine, Hilary Snaith formerly of The Salk Institute and now at the University of Edinburgh, UK, and Shelley Sazer of the Baylor College of Medicine.
Related Web sites: