DNA is constantly exposed to endogenous and exogenous agents that produce lesions by modifying its nucleobases. Exogenous agents include pollutants in our air and water supply, and UV- irradiation. The presence of these lesions in DNA in vivo is associated with aging, diseases such as cancer, and other genetically based diseases. We have studied a family of DNA lesions derived from exposure of purines to oxidizing conditions, known as the formamidopyrimidines (FapydA, FapydG). We have developed methods for synthesizing oligonucleotides containing these lesions, as well their respective nucleotide triphosphates. These tools have enabled us to characterize the effects of FapydA and FapydG on DNA repair and replication in vitro and in cells. Significant knowledge has been acquired concerning the biochemistry and replication in cells of these lesions. For instance, we demonstrated that FapydG is more mutagenic than 8-OxodGuo in COS-7 cells. However, there is still much to learn, particularly about FapydG, which is such a potent mutagen. Advances in our understanding of the biological effects of FapydG are hampered by the difficulty in obtaining sufficient quantities of oligonucleotides containing the lesion at defined positions with no limitations on sequence. We propose to vastly improve how FapydG containing oligonucleotides are chemically synthesized (Aim 1). Successful implementation of this strategy will make oligonucleotides containing FapydG more readily available for the community as a whole. We also request support to advance studies of the effects of the FapydG and FapydGTP DNA polymerase and repair enzymes. We have 2 general goals during the requested funding period. 1. Examine the mutagenicity of FapydG in mammalian cells (Aim 4), as well as the interactions of FapydG and FapydGTP with mammalian nuclear enzymes in the test tube (Aims 2 and 3). 2. Obtain the first structural information on FapydG (as opposed to analogues) and FapydGTP using x-ray crystallography. We have assembled a team of expert collaborators (Professors Basu, David, and Freudenthal) to work with us on this project. In summary, the project combines organic chemistry, biochemistry, and molecular and cellular biology to increase our understanding of fundamentally important chemical processes that occur in living organisms, and potentially improve human health. Specifically, increased understanding of the effects of the FapydG on nucleic acid structure and function will be useful for understanding the association between nucleic acid damage and the etiology of diseases such as cancer, as well as aging.

Public Health Relevance

to public health: Nucleic acid damage has a significant impact on human health. Damaged DNA is associated with aging and a variety of diseases, such as cancer. Human DNA is damaged by a variety of endogenous and exogenous agents. Examples of the latter include pollutants and UV-irradiation. Understanding how damaged DNA affects cellular processes, such as replication enhances our molecular level understanding of the etiology of diseases, as well as the various treatments for which nucleic acids are the target.

National Institute of Health (NIH)
National Institute of Environmental Health Sciences (NIEHS)
Research Project (R01)
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Cancer Etiology Study Section (CE)
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Shaughnessy, Daniel
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Johns Hopkins University
Schools of Arts and Sciences
United States
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Basu, Ashis K (2018) DNA Damage, Mutagenesis and Cancer. Int J Mol Sci 19: