DNA is constantly exposed to damaging agents. These agents may be endogenous, the products of normal cellular metabolism, or exogenous, such as ultraviolet light or chemicals. Unrepaired damage may be lethal to the cell or may cause mutations, leading to various diseases including cancer. All cells, from single-celled organisms to those in complex human organ systems, have scanning and repair mechanisms to minimize such DNA damage.
Most of the insight into how DNA scanning and repair systems work has been accomplished using prokaryotic organisms. In virtually all instances, similar systems appear in higher organisms, indicating the importance of the systems and justifying the use of prokaryotes as models of DNA repair. "Many types of DNA damage have been discovered along with many specific repair systems," says Roel M. Schaaper, a visiting scientist at the NIEHS.
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Roel M. Schaaper
Photo Credit: Steve McCaw, Image Associates
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Miriam Sander
Photo Credit: Steve McCaw, Image Associates
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To understand the various pathways and to determine how efficient the DNA repair systems are, scientists must study all the processes involved. In the future such research could lead to better treatment and prevention of cancer and other diseases. Researchers at the NIEHS's Laboratory of Molecular Genetics are seeking to understand DNA repair at the molecular level.
Molecular Genetics Research
Senior staff fellow Miriam Sander investigates base excision repair--the cell's first line of defense against oxidative and alkylative types of damage that occur frequently and accumulate during normal metabolism.
Sander is currently studying a repair enzyme, called Rrp1, that is present in the fruitfly and appears to play a role in several types of oxidative and alkylative damage. The enzyme has AP endonuclease activity that repairs either apurinic or apyrimidinic sites where DNA damage has led to the loss of a nucleotide base. When AP activity is missing in
Escherichia coli
bacteria or yeast cells, they are more sensitive to DNA-damaging agents such as radiation, alkylating agents, and hydrogen peroxide. Sander demonstrated that when Rrp1 is introduced in these repair-deficient cells, they are protected.
Sander's lab has created a fruitfly model that produces elevated levels of Rrp1. The somatic mutation and recombination rate is altered in these flies, as detected by changes in the eye pigment. This model will enable researchers to study the repair of different types of DNA damage in fruitflies with variable capacity for DNA repair.
Sander plans to engineer fruitflies with deficient repair systems, which could lead to an understanding of how a deficiency in Rrp1 affects mutation rates or even the life span of fruitflies. Although no human disease states are currently associated with this type of defect, Sander and others hypothesize that future studies may show a connection.
Post-Replication Mismatch Repair
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Thomas A. Kunkel
Photo Credit: Steve McCaw, Image Associates
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The process of DNA replication is another source of mutations. Research geneticist Thomas A. Kunkel and the members of his laboratory use human cells to study post-replication mismatch repair. The repair of replication errors can be thought of as the spell check function on a word processor. This process ensures nucleotide bases are appropriately paired. Inactivation of post-replication repair is associated with the majority of cases of hereditary nonpolyposis colorectal cancer (HNPCC). The repair defect may also account for a significant percentage of the 20,000 cases of colon cancer in the United States every year, as well as play a role in other cancers. "When the genes responsible for HNPCC were identified, they provided a molecular hypothesis for cancer," Kunkel says. "However, just saying that a defect in mismatch repair is responsible for a hereditary cancer is years away from understanding why that cell becomes cancerous."
Kunkel is trying to identify the genes involved in this type of repair and to understand their roles. As more genes are discovered, screening tests can be developed to better identify individuals who have inherited the mutation and may be at higher risk of developing the associated cancers. This research could also improve treatment strategies. New chemotherapies may be created that are more selective for specific cancer cells while sparing noncancerous cells.
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Leroy Worth, Jr.
Photo Credit: Steve McCaw, Image Associates
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Senior staff fellow Leroy Worth, Jr. studies a different aspect of post-replication mismatch repair. Using
E. coli, he investigates the enzymology behind the role of mismatch repair in controlling homologous recombination, the erroneous recombination of similar but not identical DNA sequences.
The process of methyl-directed mismatch repair in
E. coli
involves 10 different proteins. Worth determined that two of these, MutL and MutS, block the exchange of DNA between strands that are 3% different from each other. "Adding MutS only slows down the efficiency of the process; some exchange is still allowed," Worth says. To fully block the process, MutL was needed. This finding is consistent with models for how these proteins behave in replication fidelity. Although
E. coli
possess only one MutS gene, higher organisms have multiple MutS homologues, or similar genes, that may play different roles in genome stability. Worth plans to study the role these homologues have in recombination in higher cells.
Schaaper investigates strains of
E. coli
defective in the mismatch repair system. His lab inactivated mismatch repair in
E. coli
and compared the new strains to unaltered, wild-type strains that naturally have low mutation levels. The investigators observed significantly increased mutation levels (between 100- to 1000-fold) in the repair-deficient strain. In another strain, the investigators inactivated both the mismatch repair system and the proofreading system, yielding an exceptionally strong mutator strain (10,000- to 100,000-fold increase). These studies have provided detailed information about the efficiency of both the mismatch repair and proofreading systems.
Antimutators
Schaaper also studies what causes spontaneous mutations. "This is a fundamental question," he says, "because we know that spontaneous mutations are associated with birth defects, cancer, and perhaps some types of heart disease."
To address this question, Schaaper's lab created several strains of
E. coli
that produce significantly fewer errors in DNA replication than the wild-type. These "antimutator" strains possess an altered form of the DNA polymerase that is responsible for the accuracy of chromosomal replication. The effect on the replication accuracy depends on the test system, but generally varies between two- and threefold.
Schaaper determined that in the strains with the increased replication accuracy, the level of spontaneous mutations is reduced by twofold. "Such research provides a clear indication that mutation rates are genetically controlled and that an organism can change its mutation rate," he says.
The twofold decrease suggests that as much as one-half of the total mutations in a normal cell could be caused by uncorrected DNA replication errors. Schaaper speculates that the other half could be caused by DNA damage that escapes the DNA repair systems. He and his colleagues are working to extend their antimutator approach to examine the effects of various types of DNA damage, such as oxidative damage resulting from the cell's internal metabolism. "If we can demonstrate that increased levels of protective enzymes lead to decreases in spontaneous mutations, then we would have the most direct evidence that such damage contributes to spontaneous mutations," he states. While the information is not directly applicable to humans, it should greatly facilitate the identification of similar processes in higher organisms, Schaaper says.
Double-Strand Break Repair
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Micahel Resnick
Photo Credit: Steve McCaw, Image Associates
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A completely different type of repair is being investigated by research geneticist Michael Resnick. He studies the repair of DNA double-strand breaks. These breaks are unique, as DNA damage typically occurs on only one strand of the double-helix molecule, providing continuity of the genetic information as the other strand remains intact. When a DNA double-strand break occurs, there is no scaffold to hold the DNA together, and it breaks apart. "This is the basis for chromosomal breaks," Resnick says. "There are DNA breaks measured at the molecular level and chromosomal breaks at the cell level" and the latter may lead to chromosomal rearrangements and changes, he says.
Resnick discovered the recombinational mechanism of double-strand break repair in yeast more than 20 years ago. Other investigators have found human homologues for genes involved in this repair, although their role is undetermined. From yeast studies, Resnick demonstrated unrepaired DNA double-strand breaks leads to cell death. "We want to identify genes involved in turning this into a lethal event," he says. Resnick and his lab want to identify chemical agents that enhance or modify the response. The yeast model is also useful for examining DNA lesions in general, allowing investigators to understand how cells respond to damage and how to control the process.
In separate research funded in part by the National Center for Human Genome Research, Resnick's group developed a novel approach to cloning DNA that greatly increases the quality of isolated human DNA compared to other techniques. The technique, transformation-associated recombination, uses double-strand break repair and is rapid, simple, and can be used to isolate specific DNAs. Resnick says that the technique should help speed up the isolation of complete human genes and may be useful for gene therapy studies.
Barbara Proujan
Understanding how DNA replicates itself, how errors occur during replication, and how these errors are sometimes repaired are keys to unlocking potential therapies for many kinds of cancer and perhaps the secrets to the vulnerability of HIV. In this year's G. Burroughs Mider Lecture, titled "DNA Replication Fidelity, Mismatch Repair, and Genome Stability," Thomas A. Kunkel, a research geneticist in the Laboratory of Molecular Genetics at the NIEHS, presented new research on these complex topics.
The G. Burroughs Mider Lectureship was established in 1968 by the NIH scientific directors to honor Mider for his distinguished NIH career including service as director of laboratories and clinics. The award is made annually to a scientist who has contributed to the biomedical research eminence of NIH.
Kunkel's research has provided some of the greatest understandings to date of how fidelity is achieved during DNA replication. In earlier years, analyses of the molecular basis of mutation were necessarily based on solely genetic measurements. Next, simplistic
in vitro
measurements were pursued. Finally, a system was developed that monitors the products of DNA synthesis
in vitro
using sophisticated reporter target molecules. This system, developed by Kunkel and the basis for much of the research presented in his lecture, and similar systems and extensions are now in use throughout the world.
As an adjunct to this system, Kunkel developed a means of recovering engineered DNA sequences with an efficiency so great that it made phenotypic screening unnecessary. This process has also come into widespread use, and its patent is a significant source of income for the NIH and for Kunkel's laboratory.
Kunkel has also made numerous other research contributions including uncovering a general mechanism of mutation in which both base mispairing and template slippage conspire to produce a mutation, which can consist of either a base pair substitution or a gain or loss of a base. He named this process "dislocation mutagenesis" and showed that it contributes significantly to the spectrum of spontaneous mutations. Kunkel also conducted an extended and continuing analysis of the reverse transcriptase of the human immunodeficiency virus. He showed this enzyme to be extraordinarily inaccurate, thereby explaining the basis for the rapid evolution of the virus in its host, and suggesting new points of therapeutic attack on AIDS. Over the past several years, Kunkel's group has developed a system to measure not only polymerase fidelity but also mismatch repair capacity in human cell extracts. Using this system, they have greatly extended the understanding of how mutations serve as progenitors of cancer.
Now that the structures of polymerases have been identified by crystallographers, Kunkel has established a number of collaborations to probe the molecular basis of polymerase fidelity. These structures predict numerous contacts between atoms of the polymerase and atoms of the DNA template and primer strands. Such contacts have spurred experiments that are already revealing the chemical mechanism of condensation and are just beginning to reveal the contacts that may be critical for fidelity. Engineered changes in the polymerases are now providing information about the control of fidelity by amino acid residues, not only at the active site but also far removed.
Kunkel joined the NIEHS as a senior staff fellow in 1982 and achieved tenure in 1986, only nine years after obtaining his doctorate degree. During this time he has trained numerous postdoctoral fellows who now occupy positions in academia around the country. "He has proved to be an outstanding mentor, giving full credit where due and closely promoting the personal and professional growth of his laboratory family," said John Drake, chief of the Laboratory of Molecular Genetics.
Ronald Melnick, a toxicologist at the NIEHS, is serving a one-year assignment as an agency representative in the Environment Division of the White House Office of Science and Technology Policy, which has a primary role in advising the President and coordinating all science and technology policies and programs within the federal government.
Melnick is a group leader of the pharmacological and biomedical modeling group in the Laboratory of Quantitative and Computational Biology at the NIEHS. In his new assignment, Melnick works with the risk assessment subcommittee of the National Science and Technology Council's Committee on the Environment and Natural Resources (CENR) to develop and implement an intergovernmental risk assessment research strategy. The strategy sets priorities and coordinates federal research to strengthen the scientific basis of environmental risk assessment to more effectively guide public health decisions.
Working with the CENR, Melnick has had a major role in the preparation of an interagency assessment of potential health risks associated with oxygenated gasoline. "The NIEHS is especially glad to have Dr. Melnick serving in this role. It is most important that the research that the institute is doing, and the expertise of its staff, be applied to these major policy questions in a timely way," said Kenneth Olden, director of NIEHS.
"We're not an ivory tower operation," said NIEHS director Kenneth Olden, recently describing the mission of his institute. "We're not doing science for science's sake, or for intellectual stimulation." Olden points out that the central mission of the NIEHS is research and training for prevention and intervention of environmentally related disease and dysfunction. "A key part of that," he emphasizes, "is communicating our results to the American people."
Olden says that the NIEHS is concerned with moving research results from the laboratory to the bedside for clinical applications, to the work site, or to public health policy forums as quickly as possible. "Unless information from our laboratories and our scientists is accessible to a wider public, NIEHS cannot fulfill its mission," he said. To that end, the NIEHS has worked to develop a number of communication channels to ensure that the public, the scientific community, and federal agencies have ready access to information on environmental health.
People with questions about air pollution, lead toxicity, Lyme disease, radon, drinking water, and scores of other subjects can contact EnviroHealth, a clearinghouse of information dedicated to providing answers to these questions. Since beginning operations in October 1994, Envirohealth (operated by Information Ventures, Inc., and funded by the NIEHS) has fielded 3,485 calls, and in one month alone mailed out 1,176 fact sheets to callers. Calls have come from as far away as Germany, the United Kingdom, and New Zealand. People inside the United States can access EnviroHealth by calling a toll free number, mailing an inquiry, or by using electronic mail. Typical questions are Does environment play a role in breast cancer? What are the health effects of dioxin and where does it come from? and What is known about the health effects of electromagnetic fields from power lines and from home electrical appliances such as electric blankets? If the staff at EnviroHealth don't know the answer, they try to provide the caller with access to someone who does.
People with access to the Internet can scan the NIEHS gopher server located at gopher.niehs.nih.gov, and the NIEHS homepage located at URL: http://www.niehs.nih.gov on the World Wide Web for information on a variety of institute research programs and publications (see
EHPnet).
Detailed information about the National Toxicology Program, its research, and programs is available from the NTP Liaison Office at the NIEHS. The Central Data Management office of the NIEHS also provides copies of NTP Technical Reports on hundreds of animal studies on chemicals of environmental concern, as well as copies of the
Biennial Report on Carcinogens, a Congressionally mandated publication.
Information about NIEHS research and training grants can be obtained from the Division of Extramural Research and Training. Any questions not answered through one of the above pathways may also be directed to the NIEHS Office of Communications. "We serve the entire American public," Olden said. "Physicians, public health officials, educators, regulators, industry, the media, scientists . . . we are here for virtually everyone with an environmental question or concern."
Breaking into the Field
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Advancing achievement.
The Division of Extramural Research and Training is encouraging minority scientists to enter careers in environmental health.
Photo Credit: NIEHS
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Taking a creative approach to fostering the careers of minority scientists was one of the recommendations of an NIEHS-sponsored task force on the advancement of minorities in science. The NIEHS Division of Extramural Research and Training (DERT) responded to this recommendation recently with an innovative workshop format, which provided a hands-on learning environment for a dozen young minority doctorates and addressed many of the career challenges facing young investigators today.
Scientists from universities around the country who are supported by NIEHS grants received two days of intensive sessions on August 28 and 29 at the NIEHS that focused on career opportunities in environmental health sciences, applying for research grants through the National Institutes of Health, positions and funding available through the private sector, and related topics of immediate concern to post-doctoral professionals.
In her keynote remarks, DERT Director Anne Sassaman related the NIEHS training goal of attracting competitive minority scientists to careers in environmental health science to address the health problems that may disproportionately affect minorities and lower socioeconomic groups. The workshop was seen by NIEHS officials as a critical first step in achieving that goal. NIEHS Director Kenneth Olden encouraged the scientists to enjoy and pursue knowledge in the best institutions and research environments and to spend as many as five years to establish their research credentials.
The NIEHS workshop was a departure from the traditional didactic approach used to instruct young scientists in grant writing and was designed around a framework of DERT staff and extramural scientists working one-on-one with the participants to help them develop their research proposal skills. Participants were required to submit a skeleton grant application. Each participant then had the opportunity to apply instructions and guidance given to them by senior extramural scientists, industry representatives, and expert consultants to their own research projects and to receive immediate feedback on their applications. Team presentations by DERT staff highlighted specific examples of good and bad hypotheses, development of specific aims, and how general directions about writing research proposals are applied in "real life" situations. Presentations by outside experts from such organizations as Procter & Gamble, Weinberg Consulting Group, and the National Institute of General Medicine explained details of how industry and government handle grant applications.
Due to the success of this workshop and the positive reaction of the participants to the new approach, the NIEHS is planning to continue them. The ultimate success of this workshop, however, will be judged by the success of these young scientists and increases in the numbers of minority scientists contributing to the field of environmental health.
Last Update: May 1, 1997