DNA is constantly exposed to endogenous and exogenous agents that produce lesions by modifying its nucleobases. The presence of these lesions in DNA in vivo is associated with aging, diseases such as cancer, and other genetically based diseases. However, DNA damage can also be beneficial. For instance, ionizing radiation is the most common nonsurgical method used to treat cancer. Radiation kills tumor cells by damaging DNA. The goals of this research are to understand how lesions produced in DNA as a result of oxidative stress are repaired, replicated, and react to form other lesions. Our effort is focused on oxidized abasic lesions, which are incapable of forming Watson- Crick hydrogen bonds. Contrary to what was previously believed, oxidized abasic lesions interact with polymerases in distinct ways from each other and from an abasic site (AP) resulting from formal hydrolysis of a nucleotide's glycosidic bond. Hence, the inability to form Watson- Crick hydrogen bonds does not mean that a lesion is noninstructive. We will use synthetic chemistry to synthesize lesions, rapid-quench kinetics to determine polymerase mechanisms, mechanistic studies to determine irreversible inhibition of repair, macromolecular NMR to determine structure, and carry out mutagenesis experiments in yeast to determine the properties of the individual lesions. In addition, we will investigate how oxidized abasic lesions react in nucleosomes. Do nucleosomes affect the reactivity of the lesions? Do they form DNA-protein cross-links? Studies in nucleosomes bring us one step closer to studying DNA damage in cells. We will also design methods for studying DNA repair of abasic sites in cells by using photoacids in conjunction with laser photolysis to produce the lesions. We are also developing modified nucleotides that will be irreversible inhibitors of DNA polymerase b, and important enzyme in base excision repair that is over expressed in tumor cells. In summary, the project combines organic chemistry, biochemistry, and biology with the goal of improving fundamentally important chemical processes that occur in living organisms.

Public Health Relevance

DNA damage and repair are important chemical processes that significantly impact human health. These chemical processes are associated with aging and a variety of diseases, such as cancer. Understanding the chemistry, biochemistry, and biological effects of damaged DNA enhances our molecular level understanding of the etiology of diseases, such as cancer, as well as the various treatments for which nucleic acids are the target.

National Institute of Health (NIH)
National Institute of General Medical Sciences (NIGMS)
Research Project (R01)
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Synthetic and Biological Chemistry A Study Section (SBCA)
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Fabian, Miles
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Johns Hopkins University
Schools of Arts and Sciences
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Laverty, Daniel J; Averill, April M; DoubliƩ, Sylvie et al. (2017) The A-Rule and Deletion Formation During Abasic and Oxidized Abasic Site Bypass by DNA Polymerase ?. ACS Chem Biol 12:1584-1592
Zhang, Yingqian; Zhou, Xiaoping; Xie, Yonghui et al. (2017) Thiol Specific and Tracelessly Removable Bioconjugation via Michael Addition to 5-Methylene Pyrrolones. J Am Chem Soc 139:6146-6151
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Hida, Naoki; Aboukilila, Mohamed Y; Burow, Dana A et al. (2017) EC-tagging allows cell type-specific RNA analysis. Nucleic Acids Res 45:e138
Laverty, Daniel J; Greenberg, Marc M (2017) In Vitro Bypass of Thymidine Glycol by DNA Polymerase ? Forms Sequence-Dependent Frameshift Mutations. Biochemistry 56:6726-6733
Li, Fengchao; Zhang, Yingqian; Bai, Jing et al. (2017) 5-Formylcytosine Yields DNA-Protein Cross-Links in Nucleosome Core Particles. J Am Chem Soc 139:10617-10620
Pidugu, Lakshmi S; Flowers, Joshua W; Coey, Christopher T et al. (2016) Structural Basis for Excision of 5-Formylcytosine by Thymine DNA Glycosylase. Biochemistry 55:6205-6208
Weng, Liwei; Greenberg, Marc M (2015) Rapid Histone-Catalyzed DNA Lesion Excision and Accompanying Protein Modification in Nucleosomes and Nucleosome Core Particles. J Am Chem Soc 137:11022-31

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