This program seeks to obtain a fundamental understanding of the physical and chemical mechanisms by which DNA repair glycosylases locate and catalytically repair damaged bases in a vast excess of undamaged base pairs in genomic DNA. The driving force for these efforts is the belief that this mechanistic knowledge can be used to rationally engineer the specificities of these enzymes for biotechnology and biomedical applications, and will also lead to the design of useful small molecule inhibitors for antiviral therapies and other applications.
The specific aims of this proposal are to (i) Establish whether uracil DNA glycosylase (UDG) locates damaged sites in DNA by a sliding or 3D search strategy. Considerable debate exists in the DNA repair field concerning the role of processive enzyme translocation along the DNA in the site location mechanism. We will perform experiments to quantify the mean translocation distance along the DNA, the lifetime of specific and nonspecific protein-DNA complexes, the relative importance of short range and long range hopping between sites, and the efficiency of transfer between two damaged sites as a function of distance between sites, (ii) Discover how UDG traps extrahelical DNA bases that arise by spontaneous base pair breathing, and how this step leads to discrimination between normal and damaged base pairs in the genome. We will employ our recently developed imino proton exchange NMR methods to dissect the thermodynamic and kinetic features of normal and altered base pairs that promote or hinder extrahelical recognition by UDG. (iii) Understand the dynamic and structural differences between nonspecific and specific UDG-DNA complexes. We will use heteronuclear NMR methods to explore the structural and dynamic differences between free UDG and its complexes with undamaged and damaged DNA. These unique solution studies should provide the basis for how UDG exquisitely discriminates between undamaged and damaged DNA. (iv) Develop cell permeable inhibitors of human UDG (hUDG) based on high affinity inhibitors that promote base flipping or transition state interactions. Removal of uracil by hUDG is a key requirement in the life cycle of HIV-1. We will design cell permeable oligonucleotide inhibitors of hUDG using already established mechanistic principles of extrahelical base recognition and transition state mimicry. ? ? ?

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
2R01GM056834-11A2
Application #
7193614
Study Section
Macromolecular Structure and Function A Study Section (MSFA)
Program Officer
Marino, Pamela
Project Start
1998-02-01
Project End
2010-08-31
Budget Start
2006-09-21
Budget End
2007-08-31
Support Year
11
Fiscal Year
2006
Total Cost
$351,794
Indirect Cost
Name
Johns Hopkins University
Department
Pharmacology
Type
Schools of Medicine
DUNS #
001910777
City
Baltimore
State
MD
Country
United States
Zip Code
21218
Weiser, Brian P; Rodriguez, Gaddiel; Cole, Philip A et al. (2018) N-terminal domain of human uracil DNA glycosylase (hUNG2) promotes targeting to uracil sites adjacent to ssDNA-dsDNA junctions. Nucleic Acids Res 46:7169-7178
Rodriguez, Gaddiel; Esadze, Alexandre; Weiser, Brian P et al. (2017) Disordered N-Terminal Domain of Human Uracil DNA Glycosylase (hUNG2) Enhances DNA Translocation. ACS Chem Biol 12:2260-2263
Weiser, Brian P; Stivers, James T; Cole, Philip A (2017) Investigation of N-Terminal Phospho-Regulation of Uracil DNA Glycosylase Using Protein Semisynthesis. Biophys J 113:393-401
Esadze, Alexandre; Rodriguez, Gaddiel; Weiser, Brian P et al. (2017) Measurement of nanoscale DNA translocation by uracil DNA glycosylase in human cells. Nucleic Acids Res 45:12413-12424
Esadze, Alexandre; Rodriguez, Gaddiel; Cravens, Shannen L et al. (2017) AP-Endonuclease 1 Accelerates Turnover of Human 8-Oxoguanine DNA Glycosylase by Preventing Retrograde Binding to the Abasic-Site Product. Biochemistry 56:1974-1986
Seamon, Kyle J; Bumpus, Namandjé N; Stivers, James T (2016) Single-Stranded Nucleic Acids Bind to the Tetramer Interface of SAMHD1 and Prevent Formation of the Catalytic Homotetramer. Biochemistry 55:6087-6099
Cravens, Shannen L; Stivers, James T (2016) Comparative Effects of Ions, Molecular Crowding, and Bulk DNA on the Damage Search Mechanisms of hOGG1 and hUNG. Biochemistry 55:5230-42
Hansen, Erik C; Ransom, Monica; Hesselberth, Jay R et al. (2016) Diverse fates of uracilated HIV-1 DNA during infection of myeloid lineage cells. Elife 5:
Cravens, Shannen L; Schonhoft, Joseph D; Rowland, Meng M et al. (2015) Molecular crowding enhances facilitated diffusion of two human DNA glycosylases. Nucleic Acids Res 43:4087-97
Seamon, Kyle J; Sun, Zhiqiang; Shlyakhtenko, Luda S et al. (2015) SAMHD1 is a single-stranded nucleic acid binding protein with no active site-associated nuclease activity. Nucleic Acids Res 43:6486-99

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