DNA: A Sloppier Copier

University researchers have resolved an enigma of more than three decades standing by definitively establishing how ultraviolet radiation causes genetic mutation in a common bacteria.

The resolution is a surprise. While radiation is the stimulus, the group found, most of the resulting mutations are self-inflicted wounds, caused by a highly error-prone emergency DNA copying system of a novel kind.

The discovery of this system, announced in a paper in the Aug. 3 issue of the Proceedings of the National Academy of Sciences, sheds light on a fundamental cell survival process. The process is highly significant in aging, cancer and species evolution. It may even offer a clue to the solution of another long-standing mystery -- why the immune system's globulin genes are so prone to error.

A research team led by Myron F. Goodman, Ph.D., of the University of Southern California, studied a complex of evanescent substances seen at sites of DNA replication in Escherichia coli bacteria exposed to ultraviolet light. The team has established that the complex is a DNA polymerase, the fifth DNA replicating enzyme to be identified in the organism.

The new enzyme, named "pol V," differs in two significant ways from pols I, II and III, which are well-known enzymes.

First, pol V is a highly inaccurate replicator of DNA. While the other three enzyme systems copy and reproduce information-carrying sequences of the chemical bases in the DNA alphabet with an error rate of fewer than one mistake per million bases, the newly discovered enzyme is about 100 times more error-prone.

Second, pol V's structure is quite distinct from the others', which are all variations on a single design first identified in 1956 by Arthur Kornberg in work for which he shared the 1959 Nobel Prize in medicine. Dr. Goodman, a professor of biological sciences in the USC College of Letters, Arts and Sciences, notes that discovery of pol V stems from a long series of discoveries that date back almost to the beginnings of modern molecular genetics.

It is widely accepted in science that exposure to ultraviolet light and other stresses, including certain oxidizing chemicals, can create mutations -- changes in genetic make-up. Scientists studying bacteria's response to such radiation found that these organism have a highly developed emergency defense mechanism to repair damage caused by radiation. Within an hour after exposure, enzymes begin repairing damaged DNA.

"The cell has an extraordinary ability to repair and restore damaged DNA," Goodman says. "So the mystery was, if these repair systems were so efficient, why did ultraviolet light do so much damage? Why did we see so many mutations?"

Intensive research revealed that the DNA-replication mechanism became inaccurate only when a complex of unusual proteins -- given the name umuC and umuD -- were present. A special "locked" section of the bacteria's DNA produces these umu proteins, Goodman explains. Under certain stresses, including ultraviolet radiation, a cell protein called RecA unlocks that section, permitting production of the umu proteins in what is called the "SOS response."

"What everyone assumed," says Goodman, "was that these umu proteins somehow inhibited, or interfered with, the DNA copying enzymes and somehow lowered the copying fidelity of pol III, the enzyme thought to be doing the copying in SOS conditions."

But it proved frustratingly difficult to determine how that reduction in copying fidelity occurred. The umuC protein posed such extraordinary technical problems of purification that one researcher, comparing the research challenge to slopes reserved for expert skiers, called it the "black diamond of DNA biochemistry."

In the latest experiment, however, Goodman and his colleagues succeeded in elucidating this protein complex, which has been designated umuD'2C. It consists of two slightly modified molecules of the umuD protein, plus one molecule of the umuC protein.

Rather than merely interfering with the activity of pol III, this protein was a DNA polymerase in its own right -- and one of a novel form. Combined with RecA, unuD'2CC functions as a highly efficient copier of ultraviolet-damaged DNA. It is, in fact, about 100 times as efficient as pol III.

It is also, however, far more inaccurate, making mistakes about 100 times more often. Not only does it reproduce the errors caused by ultraviolet light, but it introduces errors of its own.

"What this seems to be," says Goodman, "is a last-ditch cell defense. Faced with a choice between possible mutation and death, the cell chooses possible mutation."

If so, the evolutionary consequences are obvious. "Exposing a colony of bacteria to stress provides a mechanism that causes mutations that may produce organisms more suited to the changed environment," Goodman says.

Based on earlier reports from Goodman's laboratory (strongly suggesting but not yet proving that the umu complex was a polymerase), other labs have already found analogous new polymerases that act as DNA copiers in humans.

Goodman says the discovery of an inaccurate DNA copying system immediately suggests a possible tie-in to one of the most mysterious aspects of the human immune system. The globulin gene, which controls the production of B-cells, is notoriously mutation prone.

This propensity, called "somatic hypermutability," is a crucial part of the effectiveness of the immune system's B-cells. These cells produce antibodies called immunoglobulins in a bewilderingly "hypervariable" variety of configurations.

This wild-card feature has been traced to an area, called the "hypervariable" region, on the B-cell's immunoglobin-producing genes; but the mechanism by which this hypervariability is achieved has thus far remained completely mysterious, with the most recent explanation (a problem in a repair mechanism) recently disproved.

"But an inaccurate copier, like the one we found in E. coli, could produce precisely the effects we observe," says Goodman, predicting that experiments will soon test this hypothesis.

The pol V research was supported by grants from the National Institutes of Health.

Besides Goodman, members of the research team were graduate students Xuan Shen and Mengjia Tang of the USC College of Letters, Arts and Sciences; Ekaterina G. Frank and Roger Woodgate of the NIH Section on DNA Replication, Repair and Mutagenesis; and Mike O'Donnell of Rockefeller University and the Howard Hughes Medical Institute.