The broad objective of this proposal is to understand, at the molecular level, an important defense mechanism against potentially lethal, mutagenic and/or carcinogenic insults to DNA in human cells. Such insults result from a wide variety of known environmental agents. Our approach has been to examine the role of chromatin structure in the process of recognition and removal of DNA lesions by repair enzymes in normal and (partially) repair deficient human cells. In the present application, I have proposed to examine the efficiency of DNA repair within different subdomains of nucleosomes in both human cells and a model yeast minichromosome. We will use novel exonuclease mapping techniques to determine the distribution of repair synthesis and DNA lesions following different extents of repair. These studies should indicate if certain regions of nucleosomes are refractory to efficient repair, and if repair deficient human and yeast cells lack factors required for efficient repair in the various nucleosome subdomains. We will also examine nucleosome rearrangements associated with repair of lesions induced by """"""""short patch"""""""" agents (e.g., ionizing radiation and bleomycin) to determine if the reduced DNA processing during these events requires different structural rearrangements than """"""""long patch"""""""" repair. Secondly, we will use a reversible, permeable cell system to """"""""tag"""""""" newly inserted repair patches in human cells with biotinylated nucleotides. These tags will allow us to isolate newly repaired regions of chromatin for studies on their protein composition and determine the distribution of repair sites in the human genome by electron microscopy. We hope to eventually sequence and identify proteins in human cells associated with different stages of DNA repair, and which may be altered in repair deficient human cells. Thirdly, we will explore the possibility that modification of nuclear proteins plays an active role in the DNA repair response. We plan to use the biotin-tag system to determine the level of acetylation and ADP-ribosylation of histones and nonhistone proteins in newly repaired regions. Furthermore, we will examine the efficiency of DNA repair within nucleosome subdomains of chromatin regions containing acetylated histones. Finally, we will determine if nucleosome migration is facilitated by this modification event. The results of these studies should provide a clearer understanding of the role of environment agents in such important cellular responses as survival, mutagenesis, carcinogenesis and aging.

Agency
National Institute of Health (NIH)
Institute
National Institute of Environmental Health Sciences (NIEHS)
Type
Research Project (R01)
Project #
5R01ES002614-09
Application #
3249933
Study Section
Chemical Pathology Study Section (CPA)
Project Start
1980-08-01
Project End
1992-08-31
Budget Start
1988-09-01
Budget End
1989-08-31
Support Year
9
Fiscal Year
1988
Total Cost
Indirect Cost
Name
Washington State University
Department
Type
Schools of Arts and Sciences
DUNS #
041485301
City
Pullman
State
WA
Country
United States
Zip Code
99164
Rodriguez, Yesenia; Duan, Mingrui; Wyrick, John J et al. (2018) A cassette of basic amino acids in histone H2B regulates nucleosome dynamics and access to DNA damage. J Biol Chem 293:7376-7386
Brown, Alexander J; Al-Soodani, Aneesa T; Saul, Miles et al. (2018) High-Throughput Analysis of DNA Break-Induced Chromosome Rearrangements by Amplicon Sequencing. Methods Enzymol 601:111-144
Mao, Peng; Brown, Alexander J; Esaki, Shingo et al. (2018) ETS transcription factors induce a unique UV damage signature that drives recurrent mutagenesis in melanoma. Nat Commun 9:2626
Mao, Peng; Wyrick, John J; Roberts, Steven A et al. (2017) UV-Induced DNA Damage and Mutagenesis in Chromatin. Photochem Photobiol 93:216-228
Mao, Peng; Brown, Alexander J; Malc, Ewa P et al. (2017) Genome-wide maps of alkylation damage, repair, and mutagenesis in yeast reveal mechanisms of mutational heterogeneity. Genome Res 27:1674-1684
Hodges, Amelia J; Gloss, Lisa M; Wyrick, John J (2017) Residues in the Nucleosome Acidic Patch Regulate Histone Occupancy and Are Important for FACT Binding in Saccharomyces cerevisiae. Genetics 206:1339-1348
Meas, Rithy; Smerdon, Michael J (2016) Nucleosomes determine their own patch size in base excision repair. Sci Rep 6:27122
Kong, Muwen; Liu, Lili; Chen, Xuejing et al. (2016) Single-Molecule Imaging Reveals that Rad4 Employs a Dynamic DNA Damage Recognition Process. Mol Cell 64:376-387
Hinz, John M; Laughery, Marian F; Wyrick, John J (2016) Nucleosomes Selectively Inhibit Cas9 Off-target Activity at a Site Located at the Nucleosome Edge. J Biol Chem 291:24851-24856
Mao, Peng; Smerdon, Michael J; Roberts, Steven A et al. (2016) Chromosomal landscape of UV damage formation and repair at single-nucleotide resolution. Proc Natl Acad Sci U S A 113:9057-62

Showing the most recent 10 out of 34 publications