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Mutations in cells possibly due to enzyme's miscues

Lincoln, Neb. -- The duplication of DNA is as precisely choreographed as a ballet, so when University of Nebraska-Lincoln Professor of Chemistry Mark Grieb discovered that an enzyme involved in the process was tripping all over the stage in the process, he decided to find out why.

In analyzing the enzyme's protein performance, he found that the bumbling protein enzyme, called a primase, could be an important factor in natural mutagenesis, the process by which mutant genes are formed. It could even be responsible for mutations that occur in response to external factors. It also might solve another mystery, offering evidence that could show some scientists that they have long been fingering the wrong suspect in the process by which mutant bacteria are produced.

Primase is a special kind of RNA polymerase; an enzyme that initiates, or primes, DNA synthesis by initiating the synthesis of nucleic acid polymers, the building blocks of the replicated strand.
"Other nucleic acid polymerases are far less error-prone," Griep said. "Most DNA and RNA polymerases make as few as one mistake in one hundred thousand or even a million. Primase makes as many as one mistake in 20."

In the DNA replication process, the two strands of the duplex DNA are separated into two single strands. Each of the single strands has to be copied by a DNA polymerase, a process accomplished in cooperation with primase. Of the two original strands, one is called the leading strand and is more easily copied than the other, called the lagging strand. The two strands are different because their orientation is antiparallel to one another - parallel but oriented in different directions - and the leading strand just happens to be properly oriented for the DNA polymerase to work efficiently.

On the lagging strand, however, certain chemical processes involving primase have to be done over and over. Griep said that as a result of this repetitive and more complicated replication procedure, every new piece of lagging strand DNA polymer ends up connected to a short RNA polymer.

"That means even more work for the cell because you now need other enzymes to remove the RNA polymer," Griep said. "If the RNA polymer remains attached, it will cause cells to die when they divide, or at least result in mutations in the new cells."

He says that the errors that primase makes could be an important factor in natural mutagenesis, the process by which mutant genes are formed. It could even be responsible for mutations that occur in response to external factors.

Natural mutagenesis is related to "survival of the fittest" and "population adaptability," Griep said. "The molecular biological definition of 'survival of the fittest' means that a successful, or 'fit', organism leaves behind offspring who have the same sequence of DNA as the original. If the process of DNA replication creates so many errors that the offspring don't have the same DNA sequence as the parents, then the offspring won't be anything like their parents." To Griep, finding that nature uses such a magnificently error-prone enzyme like primase during DNA replication is a mystery worth solving.

It also might solve another mystery, offering evidence that could show some scientists that they have long been fingering the wrong suspect in the process by which mutant bacteria are produced.

Primase as a player in the production of mutant bacteria may prove wrong the idea held by many scientists that natural mutations are the result of rare mistakes made by DNA polymerases, Griep said. "With all the mistakes that primase makes, I see primase as a more likely cause, he said."

If primase is indeed the cause, it would point the way to understanding how genetic diversity, the result of differences in DNA sequences in a bacteria population, occurs; how some bacteria in a population survive and adapt in the face of assault by anti-bacterial medicines or other means.
By learning how primase works, it may be possible to develop drugs that act against primase so that it will kill the bacterium within which it acts and prevent that bacterium from mutating into a drug-resistant strain. By studying primase, his lab will also determine which amino acids in this enzyme are responsible for its error-prone nature.

Griep speculates that if primase plays this role in natural mutagenesis, it may be "the thing that allows evolution to happen."

"I won't go so far as to say primase is responsible for evolution. But I think it's critical for making it happen."

Griep is currently seeking an extension of his three-year grant from the National Science Foundation to undertake such studies. He also hopes that the additional funding will enable him to extend his research into the study of human primase, which is also known to be very mistake-prone.

"If human primase causes mutations in humans, then it may contribute to the initiation of cancer. That's a good reason to be concerned about it," he said.

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Griep, vice chair of the Chemistry Department, has created a zoetrope movie (No, the movie wasn't made by Director Francis Ford Coppola, who named his film producing company after the zoetrope process, in which early movies were made from photos or images.) on his web page to show the repetitive process by which DNA is duplicated. He says that his web page is visited by several people a day and that he has received many e-mail comments from students from all around the world who appreciate being able to "see" the whole process in action. The web address for the movie is