Progress 01/01/02 to 09/30/05
Outputs
Due to depletion of the ozone, living organisms are exposed to increasing levels of ultraviolet light, which generate harmful chemical changes in the genetic code, lead to errors in DNA duplication, and trigger cell death. Many organisms protect DNA against solar UV light with an enzyme called photolyase. This cancer-preventing enzyme is not found in humans. DNA photolyase can reverse the UV-induced DNA damage, which leads to skin cancer. The research goals are understanding of the reactions of photolyase and development of a photolyase treatment for human use.
Impacts
Due to depletion of the ozone, living organisms are exposed to increasing levels of ultraviolet light, which generate harmful chemical changes in the genetic code, lead to errors in DNA duplication, and trigger cell death. Many organisms protect DNA against solar UV light with an enzyme called photolyase. This cancer-preventing enzyme is not found in humans. DNA photolyase can reverse the UV-induced DNA damage, which leads to skin cancer. The research goals are understanding of the reactions of photolyase and development of a photolyase treatment for human use.
Publications
- No publications reported this period
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Progress 10/01/03 to 09/30/04
Outputs
Various organisms protect DNA against the destructive effects of solar UV light with an enzyme called photolyase. Although not found in humans, DNA photolyase is a repair enzyme that can reverse the UV-induced DNA damage, which leads to skin cancer. To date, only an outline of the mechanism of photolyase catalysis has been proposed. Important questions yet to be answered concern details of photoactivation and photorepair of photolyase. Our lab has pioneered the use of difference FT-IR spectroscopy, which enables us to identify and follow reactive moieties during normal catalysis. One major finding from our work is that, using selective isotopic labeling of tryptophan residues in bacterial photolyase, we directly observe formation of a tryptophanyl radical during DNA repair by the enzyme, the first evidence that amino acid radicals are generated during photorepair. These data offer support of a through-bond mechanism, which suggests that the details of the protein
(i.e. individual amino acid residues) are important in defining the rate and pathway for electron transfer. Secondly, we have experimentally measured the infrared spectrum of the cyclobutane pyrimidine lesion in vitro, a critical step in probing DNA structure and dynamics with vibrational spectroscopy. Difference infrared spectra permits distinct observation of carbonyl and methyl modes in the nucleic acid upon formation of the cyclobutane dimer lesion between adjacent pyrimidines. Molecular modeling allows interpretation of these experimental data supporting a modification of molecular orbitals and electrostatic surface potentials. Apparently, forward electron transfer to the cyclobutane dimer depends upon the presence of the methyl group in thymine at the C5 position.
Impacts
These basic studies benefit the human population and its agricultural activity. Our findings could lead to commercial reagents for skin cancer prevention; there have been attempts by small biotech companies to utilize DNA repair enzyme in topical skin creams as a drug for skin cancer prevention. These methods are critical for the human population. These studies positively impact the health of agricultural workers exposed to large amounts of UV light during field work. More than 90 percent of skin cancers are directly attributable to exposure to UV light. More than 100 agricultural plants are sensitive to increased UV light. Specific effects vary markedly from one species to another. Some adapt very readily while others are seriously damaged. For example, soybeans showed a 25 percent growth reduction under a simulated depletion of stratospheric ozone by 16 percent. Increased ambient UV radiation could seriously affect all agriculture. Elucidation of the basic mechanism
of this DNA repair enzyme is a basis for minimizing effects of radiation damage in people and crops.
Publications
- Raulfs, E.C. and Kim, S. (2004) Difference FT-IR investigation of photoactivation by DNA repair enzyme photolyase. Biophysical Journal 86, 246b.
- Hurley, E.K. and Kim, S. (2004) Infrared spectroscopic studies of DNA repair by photolyase. Biophysical Journal 86, 617b.
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Progress 10/01/02 to 09/30/03
Outputs
In this time period, we have achieved two goals necessary to tackle our specific research objectives. For this proposed research, biochemical and spectroscopic studies of photolyase require large quantities of highly active protein. In this progress period, we have successfully purified bacterial photolyase, with a yield of 10 milligrams per liter of culture. This protein contains the required cofactors for activity and is capable of DNA repair. In order to identify changes in the oligomer substrates that correspond to formation of a CPD lesion, it is necessary to understand the IR absorbance spectrum of thymine monomers. Spectra of crystalline thymine were obtained, and spectral features were assigned, based on analysis of model compound literature. Additionally, we have synthesized and purified the DNA lesion, a cis-syn cyclobutane dimer, in 8-base oligomers to probe the damaged DNA with vibrational techniques. The process of damaging DNA using UV light can form
many different types of lesions. The oligonucleotide sequence chosen for this study does not contain any other pyrimidines besides thymidine to eliminate TC or CC photoproducts from being formed. Even adjacent thymidine monomers can form other photoproducts, but utilization of the acetone sensitized UV irradiation method for this molecule allows only for the cis-syn CPD photoproduct to be generated. Using purified E. coli photolyase, we have confirmed that these model CPD substrates are repaired by UV-vis spectroscopy and immunoblotting. Thus, we are confident that we had generated only the molecule of interest in high purity. In effort to assign vibrational frequencies to the CPD lesion, IR spectra of the 8mer and UV8mer molecules were obtained and a difference spectrum was generated. Using the vibrational modes of thymine as a template, it can be seen that the most notable changes in the UV8mer structure correspond to the mobility of the C5 methyl group bending, stretching, and
wagging frequencies. With the formation of the cyclobutane ring, the C6 methyl groups become sterically locked in position, in relation to both in plane and out of plane motions. In the IR spectrum, there is a slight change in the region where the carbonyl groups absorb (1770 cm-1 to 1630 cm-1). This may be due to a slight shift in their stretching frequencies upon the loss of the double bond in the ring structure and the subsequent formation of the rather rigid cyclobutane ring, in essence doubling the mass of the ring moiety to which the oxygen molecules are bound. Upon recent completion of a Nd-YAG laser system for illumination, we are now poised for acquiring difference infrared data on vibrational modes from photolyase that participate in restoring monomer pyrimidines in nucleic acids. Lastly, we have generated two site-directed mutants of photolyase and, upon protein purification, will be able to probe the effects of these substitutions at the active site of the enzyme.
Impacts
Although this project is focused on basic research, the studies benefit the human population and its agricultural activity. These experiments could lead to commercial reagents for skin cancer prevention; there have been attempts by small biotech companies to utilize DNA repair enzyme in topical skin creams as a drug for skin cancer prevention. These methods are critical for the human population. These studies positively impact the health of agricultural workers, exposed to large amounts of UV light during field work. In Virginia alone, in which high altitudes result in greater UV light incidence, there were an estimated 30,500 new skin cancer cases in 2001; well over one million cases of skin cancer were expected to be diagnosed in the United States in 2001. More than 90 percent of skin cancers are directly attributable to exposure to UV light. In the year 2000, overall cost for all cancers for medical bills, lost productivity and premature morbidity is $180.2
billion. Out of 200 agricultural plants tested, more than half are sensitive to UV light increases. UV light effects vary markedly from one species to another: some adapt very readily while others are seriously damaged. For example, soybeans showed a 25 percent growth reduction under a simulated ozone depletion of 16 percent. Ozone depletion of 10 percent or more could seriously affect all agriculture, but even smaller depletions could have a severe impact on local ecosystems. Thus, the elucidation of the basic mechanism of this DNA repair enzyme is important for both crops and people in the state of Virginia.
Publications
- No publications reported this period
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Progress 10/01/01 to 09/30/02
Outputs
Various organisms protect DNA against the destructive effects of solar UV light with an enzyme called photolyase. Although this cancer-preventing enzyme is present in numerous organisms, it is not found in humans. DNA photolyase is a repair enzyme that can reverse the UV-induced DNA damage, which leads to skin cancer. The long-range research goals are the understanding of the reactions of photolyase and the development of a photolyase treatment for human use. To date, only an outline of the mechanism of photolyase catalysis has been proposed. We are exploring details of light-driven processes in photolyase, known as photoactivation and photorepair. In this time period, we have achieved two goals necessary to tackle our specific research objectives. Biochemical and spectroscopic studies of photolyase require large quantities of highly active protein. We have partially purified cyanobacterial photolyase, expressed in bacterial systems, in milligram quantities. This
protein contains the required cofactors for activity and is capable of DNA repair. Additionally, we successfully acquired a difference infrared spectrum associated with photoactivation in cyanobacterial photolyase. Based on the following preliminary data and analysis we propose difference FT-IR band assignments for several key chemical moieties in photolyase. From this evidence, we argue that our strategy and methods are sound and that we have the experimental means to successfully complete the proposed work.
Impacts
Although this proposal is centered in basic research, these studies benefit the human population and its agricultural activity. These experiments could lead to commercial reagents to be used for skin cancer prevention; there have been attempts by small biotech companies to utilize DNA repair enzyme in topical skin creams as a drug for skin cancer prevention. These methods are critical for the human population. These studies positively impact the health of agricultural workers, exposed to large amounts of UV light during field work. The elucidation of the basic mechanism of this DNA repair enzyme is important for both crops and humans.
Publications
- No publications reported this period
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