Genome integrity, which is crucial for cell survival in response to environmental and endogenous stress, depends upon the Base Excision Repair (BER) pathway proteins. BER recognizes and removes damaged or modified bases and apurinic/apymidinic (also called abasic) sites that result from toxins, oxidative stress, and repair processes. BER damage removal and backbone incision is essential to provide the 3'primer terminus for polymerase to replace the damaged base, so polymorphisms in BER proteins are linked to cancer susceptibility, and inhibition of BER proteins is being tested in clinical cancer trials. Yet, the structural biochemistry controlling base damage recognition, pathway initiation, and damage removal remains incompletely understood. We will focus on key BER enzymes that initiate and implement committed steps in BER repair pathways, show specific recognition of damaged base(s), act in removing or processing the DNA damage, and protect the repair intermediate that would itself be toxic or mutagenic to cells.
Our Aims address hypotheses on damaged DNA recognition, catalytic mechanism, and pathway coordination for these proteins that control BER pathway initiation:
Aim 1, Uracil DNA N-Glycosylase (UDG/UNG) in deaminated base recognition and removal;
Aim 2, N-glycosylase/AP-lyases in oxidized base recognition and removal;
Aim 3, Repair endonucleases in damaged DNA backbone incision. In each Aim, the experimental strategy will define the determinants for damage recognition and catalytic mechanism through biochemical, mutational, genetic experiments integrated with structural approaches including both small angle X-ray scattering (SAXS) to characterize conformation and assembly in solution and macromolecular X-ray crystallography (MX) to reveal atomic resolution information. This work will leverage the research strengths and facilities of the investigators. The Tainer laboratory builds upon extensive experience in the MX and SAXS analyses of BER and DNA repair proteins. To help address the central challenge of linking proteins to pathways, John Tainer furthermore developed and directs the SIBYLS MX and SAXS beamline at the Advanced Light Source Synchrotron. The Cunningham laboratory has extensive experience with BER biochemistry and with quantitative mutational analyses and genetic complementation tests in E. coli. Strategically chosen collaborators bring complementary protein and technical expertise to aid rigorous analyses. The proposed structural and biochemical research will test unifying hypotheses relevant to base repair enzyme activities and functions in genomic integrity, including our overall proposition that repair pathway progression and coordination is in part an emergent property of the structural chemistry for individual protein:damaged DNA complexes. Overall, this research aims to provide fundamental knowledge of BER complexes relevant to defining their roles in the regulation of genome fidelity and the mechanisms whereby loss of the functions of these coordinated complexes may lead to sensitivity to environmental toxins, to initiation of degenerative disease and cancer, and also to opportunities for therapeutic interventions.

Public Health Relevance

Genome integrity, which is crucial for cell survival in response to environmental and endogenous stress, depends upon the Base Excision Repair (BER) pathway proteins. To characterize the biologically important activities and interactions of these BER proteins, we propose to integrate biochemical, mutational, and structural techniques and to focus efforts on BER enzymes that initiate and implement committed steps of damage recognition and incision. Overall, this research will provide fundamental knowledge on BER complexes relevant to defining their roles in the regulation of genome fidelity and the mechanisms whereby loss of their functions leads to sensitivity to environmental toxins and initiation of degenerative disease and cancer.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
5R01GM046312-20
Application #
8296693
Study Section
Radiation Therapeutics and Biology Study Section (RTB)
Program Officer
Preusch, Peter C
Project Start
1991-07-01
Project End
2013-12-30
Budget Start
2012-07-01
Budget End
2013-12-30
Support Year
20
Fiscal Year
2012
Total Cost
$612,908
Indirect Cost
$221,894
Name
Lawrence Berkeley National Laboratory
Department
Biophysics
Type
Organized Research Units
DUNS #
078576738
City
Berkeley
State
CA
Country
United States
Zip Code
94720
Tsutakawa, Susan E; Lafrance-Vanasse, Julien; Tainer, John A (2014) The cutting edges in DNA repair, licensing, and fidelity: DNA and RNA repair nucleases sculpt DNA to measure twice, cut once. DNA Repair (Amst) 19:95-107
Shin, David S; Pratt, Ashley J; Tainer, John A (2014) Archaeal genome guardians give insights into eukaryotic DNA replication and damage response proteins. Archaea 2014:206735
Dalhus, Bjorn; Nilsen, Line; Korvald, Hanne et al. (2013) Sculpting of DNA at abasic sites by DNA glycosylase homolog mag2. Structure 21:154-66
Perry, J Jefferson P; Tainer, John A (2013) Developing advanced X-ray scattering methods combined with crystallography and computation. Methods 59:363-71
Tsai, Chi-Lin; Tainer, John A (2013) Probing DNA by 2-OG-dependent dioxygenase. Cell 155:1448-50
Roberts, Victoria A; Thompson, Elaine E; Pique, Michael E et al. (2013) DOT2: Macromolecular docking with improved biophysical models. J Comput Chem 34:1743-58
Brosey, Chris A; Yan, Chunli; Tsutakawa, Susan E et al. (2013) A new structural framework for integrating replication protein A into DNA processing machinery. Nucleic Acids Res 41:2313-27
Hegde, Muralidhar L; Tsutakawa, Susan E; Hegde, Pavana M et al. (2013) The disordered C-terminal domain of human DNA glycosylase NEIL1 contributes to its stability via intramolecular interactions. J Mol Biol 425:2359-71
Tsutakawa, Susan E; Shin, David S; Mol, Clifford D et al. (2013) Conserved structural chemistry for incision activity in structurally non-homologous apurinic/apyrimidinic endonuclease APE1 and endonuclease IV DNA repair enzymes. J Biol Chem 288:8445-55
Johnson, Christopher N; Spring, Alexander M; Desai, Sunil et al. (2012) DNA sequence context conceals *-anomeric lesions. J Mol Biol 416:425-37

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