May 10, 2000 Many of us saw films in school and think of DNA replication -- the duplication of the cell -- as involving a gliding double-helix that breaks apart and smoothly forms new cells as symphonic music swells in the background. But the latest scientific studies show that cell replication is in fact much different, and far more chaotic. There can be a widespread substitution of different elements of the cell, with all sorts of effects. Those effects can be helpful, as in evolutionary change, or bad, as in cells that run amok and form cancer.
A new study from University of Washington researchers shows that a DNA polymerase -- an enzyme -- commonly used for scientific study can tolerate many different mutations and remain functional. The total number of different active mutant forms discovered, 8,000, is apparently the largest library of any polymerase yet known -- and it might be the largest library of any enzyme known.
"This presents whole paradigms of evolution. The active area of these enzymes is one of the most conserved sites in nature -- that nature has ever created. What this says is that the most conserved site in nature is plastic, within the test tube. You can put in all possible mutations," says the co-author, Dr. Lawrence Loeb. Leob is director of The Joseph Gottstein Memorial Cancer Research Laboratory at the University of Washington School of Medicine and is a professor of pathology and biochemistry and director of UW's Medical Scientist Training Program.
The other co-author is Premal Patel, who carried out the studies. He is a medical scientist training program student, in the M.D./Ph.D. program at the University of Washington.
The findings are being published in the May 9 issue of the journal Proceedings of the National Academy of Sciences.
Polymerases are enzymes -- a type of protein -- that can replicate DNA. DNA forms the genes that give us our characteristics.
The polymerase involved is called Taq, the one most often used by laboratories in polymerase chain reactions (PCR), essential to genetic research and other uses. PCR takes a small amount of DNA, and expands it into a larger amount. That's important because scientists often start with a small amount of DNA, and need a larger quantity for study. So Taq is put into the test tube when medical patients receive genetic tests, or when a forensic scientist studies DNA from a crime scene. Scientists use Taq in their search for, among other things, treatments for cancer and AIDS. Patel inserted random sequences into Taq, and found that many different amino acids would substitute for others. This was different than conventional wisdom, which held that the active site of the polymerase would not tolerate change well.
"Following selection, we found that many different amino acids could be altered without affecting the function dramatically," Patel says. "We think enzymes are highly plastic."
The research may illustrate how bacteria are able to resist antibiotics or other treatment drugs. Because their active site is so mutable, it becomes more likely that the organism may develop a resistance mutation.
Meanwhile, Loeb's laboratory now has a library of 8,000 Taqs that scientists will now scrutinize for other uses. Some of these variations may have uses in biotechnology, Patel says.
Loeb's laboratory has been inserting random sequences into different enzymes for 13 years with the goal of creating gene therapies for cancer. Loeb's laboratory has experimented with inserting random sequences into other parts of the Taq -- not the part that was the subject of the recent paper. That's because that part was the most conserved in nature, and did not on its face appear susceptible to much change.
"We sort of assumed the most conservative site in nature -- all species have it -- would not be mutable, so we avoided it," Loeb says.
Mutations are a key to Loeb's work in cancer experimentation. Twenty-five years ago, Loeb theorized that the engine behind cancer's devastation was a mutator phenotype, in which there is an increasing rate of errors in DNA replication as a tumor grows. Only within the last few years have experiments shown Loeb's theory was correct. His goal now is to find ways to slow the mutation rate. This would not cure cancer, but it would likely extend the lifetimes of most people who get it. The hope, then, would be to eliminate most cancer deaths through delay.
"People tend to think of mutations as constant -- as if you get a certain level a year," Loeb says. "You can manipulate the rates of mutation. In the case of cancer, the goal is to delay the rate of mutation. In the case of Taq, the goal is to increase mutations -- to put mutations in a cell, in the test tube, and then take advantage of the situation."
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