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Environmental Factor, June 2012

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Council talks focus on DNA repair

By Heather King

Leona Samson, Ph.D.

Samson is one of just two NIEHS-funded researchers who have won the coveted Pioneer Award, which is designed to support individual scientists of exceptional creativity who propose pioneering, and possibly transforming, approaches to major challenges in biomedical and behavioral research. (Photo courtesy of Steve McCaw)

Thomas Kunkel, Ph.D.

In 2011, NIH promoted Kunkel to the rank of NIH Distinguished Investigator, one of the highest honors NIH awards to its scientists, and one that only an estimated two to three percent of NIH scientists ever achieve. In addition to leading his research group, Kunkel is also head of the Laboratory of Structural Biology. (Photo courtesy of Steve McCaw)

The National Advisory Environmental Health Sciences (NAEHS) Council meeting May 22 at NIEHS featured scientific talks by Massachusetts Institute of Technology professor and Pioneer Award winner Leona Samson, Ph.D., and head of the NIEHS DNA Replication Fidelity Group Thomas Kunkel, Ph.D. Both talks addressed the importance of DNA repair in determining the ultimate effects of environmental exposures, from ultraviolet (UV) light to heavy metals, on human health.

The ability to correct mistakes, or lesions, in DNA that would otherwise lead to illness and disease varies greatly among individuals and is referred to as DNA repair capacity. Differences in repair capacity help explain why some individuals are more likely to fall ill from chemical exposures or develop skin cancer from sun damage, while others remain healthy. 

Samson's pioneering methods to analyze DNA repair capacity

Samson began her talk, “Developing Novel Methods to Measure DNA Repair Capacity in Humans,” by describing different types of DNA damage and the different biological repair strategies that specifically address them. One of the better-defined repair processes, nucleotide excision repair (NER), is important in correcting DNA damage by ultraviolet (UV) rays and is disrupted in the disease xeroderma pigmentosa (XP). As DNA lesions from UV exposure build up, XP patients acquire numerous skin malignancies from an early age.

Early methods for quantifying DNA repair capacity relied on expressing a reporter gene from a plasmid that contains UV-induced damage that is known to inhibit mRNA transcription. Reporter expression was robust in normal cell lines that can repair the damage, but very poor in XP cell lines that are deficient in repair. The protein produced by the reporter could then be detected based on its activity in enzyme assays, allowing researchers to quantify the amount of repair taking place. 

Going from one channel to five

Using the host cells' ability to reactivate the expression of a reporter gene in a repair-dependent manner is also the basis of Samson's new system for measuring DNA repair capacity, which is capable of tracking multiple DNA repair pathways simultaneously with an easy to read output. Instead of using a single reporter, however, Samson's system uses different shades of fluorescent proteins.

When repair occurs at a plasmid with one type of lesion, a particular shade of light can be emitted by its fluorescent protein. This system allows researchers in her lab to monitor a variety of reporters and repair pathways simultaneously with a simple fluorescent light detector.

Using this system, Samson's group has been able to evaluate DNA repair systems including NER, homologous recombination, mismatch repair, and MGMT direct reversal repair. Samson also spoke of the possibility of using these repair capacity reporting plasmids in cell lines derived from patients, which would allow a person's DNA repair capabilities and deficiencies for specific types of damage to be determined.

The bigger picture

Samson is now looking forward to using her fluorescent reporter system as a guide to direct more detailed studies of other kinds of DNA damage that do not inhibit transcription. By using high throughput sequencing technology, Samson can see how damage and repair affect transcriptional mutagenesis wherein the damaged DNA template encodes mRNAs with inappropriate sequences that may not make functional proteins.

Kunkel drives home the challenge of DNA replication fidelity

Kunkel began his talk, “The consequences of DNA replication infidelity in human health,” by drawing the audience in with a metaphor for the impressive biology that lets healthy people avoid replication errors. Imagine, he said, typing 2,000 copies of a lengthy textbook with no mistakes between 8 a.m. and 4 p.m. If that text book were the human genome, the workday would represent the 8 hours of S phase. Of course, much like a typist, the DNA replication machinery relies on it versions of the backspace function, exonuclease activity, and spellcheck, mismatch repair, as well as hitting the right keys, nucelotide selectivity.

Kunkel went on to explain the importance of the complex interaction between the polymerase making the new DNA strand and each incoming nucleotide that will be added. He showed atomic resolution structures of the new nucleotide snugly fitting into its spot in a polymerase and described the different polymerases important for proper replication.

TLS polymerases allow infidelity

After describing the scrupulous fidelity of most DNA replication polymerases, Kunkel introduced the translesion synthesis (TLS) polymerases, which are less picky and more flexible in their active sites, allowing them to replicate damaged DNA. This allows TLS polymerases to correct lesions, as they do to prevent the type of UV damage seen in the XP patients also described by Samson. These more liberal polymerases also allow errors when mutagenesis is beneficial, such as during antibody production. In this manner, TLS polymerases make the new DNA more as it should be by allowing it to be less like its template.

RNA in the DNA

Kunkel also spoke about another type of mistake during replication — the insertion of RNA bases into a new strand of DNA. Ribonucleotides contain an extra oxygen atom that can result in strand cleavage and genome instability, and the incorporation of ribonucleotides leads to damage-susceptible DNA. Why, then, have Kunkel’s group and others observed such a high rate of ribo incorporation where the corresponding DNA nucleotides should be? His research and that of others suggest there may be a signaling function behind ribonucleotide incorporation. Kunkel’s lab is excited about further examining the causes for, and ultimate effects of, ribonucleotide incorporation on human health.

(Heather King, Ph.D., is an Intramural Research Training Award fellow in the NIEHS Laboratory of Structural Biology Protein Expression Core.)

Fundamental, exposure, and translational research

The study of DNA repair helps scientists understand not only the different types of DNA damage and how they occur in response to various environmental exposures, but also the critical role of damage control in human health. Just as it is sometimes better to ask for forgiveness than permission, sometimes biology finds it more efficient to allow mistakes and correct them as necessary.

Understanding what occurs during various repair processes, how these processes help humans respond to harmful exposures, and how to determine a particular patient’s risk for disease based on repair capacity fulfills several NIEHS goals, promising to continue making good returns on the investment NIEHS makes in the critical field of DNA repair research.

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