DNA repair enzymes detect damaged DNA bases in the dramatically different contexts of naked duplex DNA as well as nucleosomes and chromatin. These fascinating and essential molecular recognition processes require dynamic motions of the DNA base pairs to expose damaged bases to the enzyme, and also, facilitated diffusion of the enzyme along the DNA chain so that stretches of the DNA chain can be meticulously interrogated in a single bimolecular encounter event. A fundamental understanding of damage recognition in the contexts of naked DNA and nucleosomes requires a paradigm system that is especially amenable to study using a wide variety of biophysical tools. One such system is the uracil base excision repair pathway (UBER). This multienzyme pathway is initiated by the enzyme uracil DNA glycosylase (UNG), which efficiently locates and excises uracil bases from DNA. The biomedical relevance of UBER arises from the central role of this pathway in the adaptive immune response, in providing innate immunity against viruses, and in mediating the therapeutic effects of fluoropyrimidine anticancer drugs. In addition, aberrant UBER has been related to carcinogenesis. The characterization of the molecular nature of these interactions provides the basis for identifying new targets for biomedical intervention in the immune response, cancer therapy and viral pathogenesis. The focus of this proposal is to: (1) Elucidate the mechanism and molecular interactions that allow human UNG to execute intramolecular facilitated transfer between uracil sites in duplex DNA. Using NMR paramagnetic relaxation enhancement (PRE) methods, we will characterize transient and poorly populated binding modes of hUNG to nonspecific and specific DNA that are important for intramolecular site transfer. Based on unique structural insights, we will mutate specific residues on UNG, and make discrete perturbations of functional groups on the DNA, to uncover enzyme-DNA interactions important for intramolecular transfer. (2) Elucidate the role of nucleosomal DNA dynamics in uracil excision by hUNG. We will use newly conceived NMR and biophysical methods to explore the dynamic properties of DNA in mononucleosomes for the first time, and we will elucidate the mechanism by which UNG locates and repairs uracil sites imbedded in nucleosomal DNA. (3) Determine the mechanism and efficiency of intramolecular facilitated transfer by hUNG between uracil sites embedded in nucleosomes. The significance and mechanism of intramolecular site transfer in the context of mononucleosomes will be elucidated using novel biophysical and chemical approaches. In particular, we will discern whether the nucleosome surface provides a pathway for intramolecular transfer, whether the DNA scaffold is the primary conduit, or if site location occurs by diffusion from bulk solution.

Public Health Relevance

The biomedical relevance of uracil base excision repair (UBER) arises from the central role of this pathway in the adaptive immune response, in providing innate immunity against viruses, and in mediating the therapeutic effects of fluoropyrimidine anticancer drugs. In addition, aberrant UBER has been related to carcinogenesis. The characterization of the molecular interactions in this pathway provides the basis for identifying new targets for biomedical intervention in the immune response, cancer therapy and viral pathogenesis.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
5R01GM056834-18
Application #
8537467
Study Section
Macromolecular Structure and Function E Study Section (MSFE)
Program Officer
Marino, Pamela
Project Start
1998-02-01
Project End
2014-08-31
Budget Start
2013-09-01
Budget End
2014-08-31
Support Year
18
Fiscal Year
2013
Total Cost
$363,491
Indirect Cost
$141,850
Name
Johns Hopkins University
Department
Pharmacology
Type
Schools of Medicine
DUNS #
001910777
City
Baltimore
State
MD
Country
United States
Zip Code
21218
Hansen, Erik C; Seamon, Kyle J; Cravens, Shannen L et al. (2014) GTP activator and dNTP substrates of HIV-1 restriction factor SAMHD1 generate a long-lived activated state. Proc Natl Acad Sci U S A 111:E1843-51
Gajula, Kiran S; Huwe, Peter J; Mo, Charlie Y et al. (2014) High-throughput mutagenesis reveals functional determinants for DNA targeting by activation-induced deaminase. Nucleic Acids Res 42:9964-75
Seamon, Kyle J; Hansen, Erik C; Kadina, Anastasia P et al. (2014) Small molecule inhibition of SAMHD1 dNTPase by tetramer destabilization. J Am Chem Soc 136:9822-5
Rowland, Meng M; Schonhoft, Joseph D; McKibbin, Paige L et al. (2014) Microscopic mechanism of DNA damage searching by hOGG1. Nucleic Acids Res 42:9295-303
Schonhoft, Joseph D; Kosowicz, John G; Stivers, James T (2013) Correction to DNA translocation by human uracil DNA glycosylase: role of DNA phosphate charge. Biochemistry 52:8599
Schonhoft, Joseph D; Stivers, James T (2013) DNA translocation by human uracil DNA glycosylase: the case of single-stranded DNA and clustered uracils. Biochemistry 52:2536-44
Weil, Amy F; Ghosh, Devlina; Zhou, Yan et al. (2013) Uracil DNA glycosylase initiates degradation of HIV-1 cDNA containing misincorporated dUTP and prevents viral integration. Proc Natl Acad Sci U S A 110:E448-57
Schonhoft, Joseph D; Kosowicz, John G; Stivers, James T (2013) DNA translocation by human uracil DNA glycosylase: role of DNA phosphate charge. Biochemistry 52:2526-35
Schonhoft, Joseph D; Stivers, James T (2012) Timing facilitated site transfer of an enzyme on DNA. Nat Chem Biol 8:205-10
Parker, Jared B; Stivers, James T (2011) Dynamics of uracil and 5-fluorouracil in DNA. Biochemistry 50:612-7

Showing the most recent 10 out of 56 publications