Our cells constantly flirt with disaster: Before each division, they duplicate hundreds -- often thousands -- of DNA snippets from each chromosome. But if any snippet gets copied twice, the daughter cells will get faulty instructions and may start a buildup of errors that can cause cancer generations hence.
Scientists at the University of California, San Francisco have deciphered the long-puzzling process by which every cell regularly averts these dangers by shutting down the gene copying process as soon as one complete copy is made.
The discovery, published in the journal Nature, involves a fail-safe system of overlapping controls requiring that three separate chemical processes be reversed before the genes can be re-copied - a highly unlikely series of events, and therefore a near-perfect protection.
The molecular-level finding was made by studying the cell cycle in common brewer's yeast, an organism that has proved a powerful model for understanding how human cells function in normal and diseased states. While some of the details may differ in humans, the basic pattern of overlapping controls and the strategies used to carry them out are expected to be similar, the researchers say.
Since, as the scientists finally found, cells use three or more separate strategies to avoid over-copying genes, determining just how the process worked has proved elusive.
"We knew of several potential ways the cell machinery might prevent inappropriate re-replication, but we were repeatedly frustrated in attempts to show that any of them were actually involved," said Joachim Li, PhD, UCSF assistant professor of microbiology and immunology and senior author on the Nature paper. "We would experimentally de-activate one mechanism after another and find no effect. This is not the kind of result that helps convince granting agencies you are on the right track."
"We eventually demonstrated that not one or two, but at least three distinct controls have to be turned off simultaneously for cells to start replicating again. This is unlikely to happen by accident, so this multi-layered protection is virtually fail-safe. That's what you want when there is no room for error."
While multiple overlapping pathways are not thought to be an uncommon safeguard, few such systems have been clearly described, Li said.
In yeast, as in human cells, DNA replication is triggered in the nucleus when proteins known as replication factors position themselves at hundreds to thousands of pre-determined sites along the DNA molecule. Li's UCSF team knew that a specific set of proteins called kinases played a central role in preventing this critical process from starting more than once. But they did not know just how the kinases acted - whether, for example, they triggered the replication proteins into action or helped block their action. Nor, of course, did they know how many steps were involved.
The scientists found that the kinases act on at least three replication factors, knocking them out of commission in different ways. A kinase causes one replication factor called Mcm to get expelled from the nucleus; another, known as Cd6, is chemically degraded. A third replication protein, called ORC for "origin recognition complex," is de-activated by kinase through a still-unknown process.
Kinases are ubiquitous enzymes widely used in cells to regulate other proteins. In a step known as phosphorylation, they add a phosphate molecule to their target, changing its shape or signaling other proteins to degrade, relocate or modify it.
Only when the scientists managed to block the effects of kinases on all three replication proteins -Mcm, Cd6 and ORV - were they able to trigger DNA replication to re-start in the cell. But even this did not lead to a complete doubling of the cell's genome. About 50 percent of the genes were re-copied, the researchers report, suggesting that still more regulatory mechanisms and possibly more replication factors are involved. Li's team is actively looking for them.
"The evolution of this overlapping system of protection indicates just how crucial it is to an organism's survival to bequeath an exact complement of every gene to each generation of cells," Li says. "Failure to pass on genetic information stably can lead to cell death, or possibly to an accumulation of genetic lesions that ultimately may give rise to cancer."
If some genes are copied more than once before the cell divides, daughter cells may inherit faulty instructions to make too much of some proteins, disrupting the balance of activities needed to control cell proliferation. Excess duplication can also disrupt the proper segregation of genetic information when the cell next divides, Li said, a potential danger that the new research will allow scientists to study.
"For the first time, we have a system to clarify just what happens when replication is not controlled," he said. "The research offers a way to move beyond the theory to actual experimental analysis of how re-replication can disrupt the integrity of genetic inheritance and how the cell responds to this disruption. Eventually, we may learn how to enhance cells' abilities to protect themselves from these disruptions."
First author on the Nature paper is Van Q. Nguyen, UCSF graduate student in biochemistry and biophysics. Co-author and collaborator on the research is Carl Co, also a graduate student in the same department.
The research is funded by the American Cancer Society and the National Institutes of Health. Critical early funding came from the Searle Scholars Program, the Markey Charitable Trust, the Rita Allen Foundation and two private contributors to UCSF: Brook Byers and Henry Wheeler.
Journal
Nature