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February 2011

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Mismatch base pairing is structurally similar to correct base pairing

By Robin Arnette
February 2011

Katarzyna Bebenek, Ph.D.

Bebenek said, "Our paper is not as groundbreaking as Watson-Crick's discovery, but it is really nice to show something that was just a hypothesis, and now we actually show that it is the case." (Photo courtesy of Steve McCaw)

Lars Pedersen, Ph.D.

Pedersen noted that he was expecting to see the guanine-thymine mismatch pair in a wobble conformation similar to what has been seen in previous crystal structures, but was elated to see the base pairs fit almost perfectly. (Photo courtesy of Steve McCaw)

Tom Kunkel, Ph.D.

Kunkel found this work to be one of the most intellectually satisfying projects he's participated in because it addresses an important hypothesis on the origins of mutations proposed over a half century ago by two of the founding fathers of molecular genetics. (Photo courtesy of Steve McCaw)

When James Watson and Francis Crick published their seminal Nature papers describing the structure of the DNA double helix in 1953, they proposed that a subtle chemical change in DNA could make a mistake, or a mismatch base pair, look like a correct base pair. This hypothesis has been under investigation for more than a half century and has proven difficult to test. Now, three NIEHS investigators from the Laboratory of Structural Biology have provided strong evidence to support Watson and Crick's theory about the origin of spontaneous base substitution mutations.

Using X-ray crystallography, Katarzyna Bebenek, Ph.D., Lars Pedersen, Ph.D., and Tom Kunkel, Ph.D., have determined that human DNA polymerase can incorporate a guanine-thymine (G•T) mismatch during DNA synthesis that is structurally similar to the correct adenine-thymine (A•T) base pair. These data( Exit NIEHS, published in a recent 2011 issue of the journal PNAS (Proceedings of the National Academy of Sciences), are the first to show that during synthesis, natural DNA bases can form mismatches that have correct Watson-Crick geometry, potentially resulting in mutations. Interestingly, these mutations aren't due to the typical sources of environmental stress on DNA, such as exposure to UV light or chemical toxins. Instead, they happen spontaneously.

"Mutations underlying diseases do not necessarily require that a person have a genetic defect or be exposed to radiation or chemicals in the environment," Kunkel explained. "This study reveals how the chemical complexity of the genetic information can sometimes trick even a normal, healthy cell into making a mistake for good, in the case of evolution or the development of a healthy immune system, or bad, when the mutation results in disease."

Bebenek is a staff scientist in Kunkel's group and is first author on the paper. She said the results were exciting, not only because understanding how a DNA polymerase generates mutations may help scientists discern the cause of disease, but because DNA polymerases serve as targets of pharmaceutical agents. Therefore, understanding the mechanism by which DNA polymerases incorporate or misincorporate nucleotides may improve drug design.

She didn't know if scientists 50 years from now would consider this paper as important as Watson and Crick's ground-breaking publications, but she's sure that the data are solid. "While we were writing this paper, we produced another crystal structure with the same mispair, but the crystal grew under different conditions and had a different space group," she explained. "The confirmation of the mismatch, basically how it sits in the polymerase, was exactly the same. It convinced me that this mismatch didn't just happen in one crystal."

As an expert on X-ray crystallography, Pedersen said that when he first saw the crystal structure, he was more excited that the active site atoms were in proper position for the catalytic reaction to proceed. It was later on that he thought about the significance of the work.

Pederson said, "Although it had been predicted to be a possibility, I was surprised to see the G•T pair with Watson-Crick-like geometry. The realization of the historical implication of this structure didn't hit me right away."

Although Kunkel has been involved in dozens of important discoveries related to DNA replication, he said that this work was one of the most important things his lab had ever done. He summed up his feelings by saying, "Watson and Crick's hypothesis was a fundamental idea in molecular biology that remained unanswered for 58 years. Because we can now study DNA at the atomic level, we were able to provide strong support for an idea that had limited evidence. That's what we've answered in this PNAS paper."

Citation: Bebenek K, Pedersen LC, Kunkel TA( Exit NIEHS. 2011. Replication infidelity via a mismatch with Watson-Crick geometry. Proc Natl Acad Sci U S A; doi:10.1073/pnas.1012825108 [Online 13 January 2011].

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