DNA is composed of two strands--the famous double helix, which resembles a twisted ladder. The "rungs" of the ladder consist of pairs of four chemical bases represented by letters. T always pairs with A to make a rung; C always pairs with G. When it's time for the cell to reproduce, it must copy, or replicate, the exact sequence of bases--millions of them in every chromosome. Mutations are typos: mistakes in the sequence.

Different types of molecules absorb different wavelengths of light, and DNA happens to absorb UV light. "As a result, the bases in DNA can be excited by that energy and undergo chemical rearrangements," says Fix. UV light can cause two neighboring bases in a DNA strand to form an abnormal bond with each other. The abnormality is called a lesion, and it gums up the works when protein complexes in the cell try to replicate or transcribe the DNA.

Fortunately, cells can repair most lesions by stripping out the damaged bit of DNA and resynthesizing it. These repair mechanisms are typically "error-free," meaning that they accurately re-create the DNA.

But when there's a lot of damage, the cell's repair capacity falters. Repair mechanisms that are "error-prone" kick in: they allow the cell to keep functioning, but at the price of mutations. They sometimes substitute a C for a T, or a G for an A.

Most mutations pose no problems. But some can trigger a cascade of cellular reactions that lead to cancer or other disorders.

By exposing cells to moderate doses of UV radiation, Fix can cause enough DNA damage to readily study DNA repair and mutations. For this work he's using a harmless laboratory strain of E. coli bacteria, a common choice for molecular biology experiments.

"We know exactly what kinds of mutations can occur in our strain of E. coli, and we know the genetic sequence," he says. "We also have access to other strains with varying capacities for DNA repair. We can analyze lots of samples very quickly and collect lots of data. It's much more difficult and much more expensive to do that with human cells, so we can cover more territory.

"Many of the mechanisms that repair DNA are fundamentally the same in bacteria and humans. By analyzing these fundamental processes in E. coli, we hope to relate them to things that happen in humans. We know that UV radiation mutates human cells, and there's good evidence to suggest that the same types of [lesions] that cause cancer in human cells cause mutations in E. coli."

Fix recently was awarded a National Cancer Institute grant to correlate certain kinds of DNA lesions with certain kinds of mutations and to study their frequency. The grant, an Academic Research Enhancement Award, will allow several undergraduates to take part in the research.

By experimenting with different repair mechanisms, Fix and his students can trace a specific type of mutation back to the type of lesion that caused the trouble. They also can determine how often different types of UV-induced lesions occur (some are a hundred times more common than others) and roughly how often they tend to result in mutations. A given type of lesion may trigger mutations--may "foil" the cell's repair capacity--way out of proportion to its incidence.

Damaged by the light

"For all the years that UV light has been studied as a mutagen, it's been very difficult to make the kinds of correlations between damage and mutation that intuitively seem obvious," Fix says. "People have looked at damage distribution and compared that to mutation frequency, and found little correlation, in many cases."

That may be because scientists have overlooked a rare type of UV damage where neighboring T and A bases become bonded, he says. "Our pilot data suggest it plays an important role in mutagenesis even though it doesn't occur very often." Part of the grant project will take a closer look at these T-A lesions and their role in causing mutations.

Fix's lab will also investigate an unexpected type of mutation that they've found sometimes results from C-T lesions, a much more common type of DNA damage. And they'll investigate another oddity they've discovered about UV-related mutations.

When the cell replicates its DNA, protein complexes must "zip open" the two strands of the DNA molecule to access them. For complicated reasons, there are differences in the way the two strands are copied. "We have some preliminary data showing a fourfold difference in the frequency of particular mutations between the two strands," says Fix. He wants to know if this is a function of the cell's replication process or if UV damage occurs more often on one strand than the other--and if so, why.

Fix's work, like Blaine Bartholomew's (see part 2 of this story), is basic rather than applied science. It's directed at finding out how things work--in this case, why, where, when, and how things go wrong in our DNA. That's fundamental in both senses of the word.

"My research isn't going to cure cancer," Fix says, "but hopefully it will help someone else understand more about the process.

"That's what basic research is all about: adding to the pool of knowledge in the hope that someone will be able to use what you've done to find something else to add. Eventually the sum of knowledge can lead to applications that are very useful to all of us."

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