The broad objective of this proposal is to understand the molecular details of transcription-coupled DNA repair, an important defense mechanism against phenotypic changes induced by insults to DNA in mammalian cells. Such insults result from a wide variety of environmental agents, such as ultraviolet (UV) radiation and chemical carcinogens. Since repair of bulky chemical adducts and UV photoproducts in DNA occurs via the same mechanism (excision repair), UV radiation is used as a prototype environmental agent for most of our studies. We will examine the relationship between DNA repair efficiency, transcriptional activity and chromatin structure of two different classes in mammalian genes, and an inducible gene in yeast. The relationship of repair to RNA polymerase II (polII) expression will be examined in mouse cells containing the herpes simplex virus thymidine kinase (tk) gene fused to the mouse mammary tumor virus long terminal repeat (LTR). This """"""""LTL"""""""" construction is stably integrated into the genome of these cells and expression of the tk gene requires glucocorticoid hormone. We have shown that hormone-induced transcription of the tk gene is acutely sensitive to UV radiation and returns rapidly following efficient repair in this construct. Since UV photoproducts form preferentially in the LTR region, transcription may be blocked by UV damage to promoter elements. We will use ligation-mediated PCR to examine the yield and repair of UV photoproducts at specific sites in the LTR region required for initiation of pol II. We will also examine the effect of UV damage on the binding of hormone receptor to the LTR using a """"""""gel- shift"""""""" assay. Repair of N-methyl purines in ribosomal RNA genes (rDNA) will be examined in mouse Friend erythroleukemia cells. We have fractionated the rDNA of these cells into transcriptionally active and inactive forms of chromatin, using psoralen crosslinking, and find that EcoRI digestion of nuclei releases only the active fraction. Unlike pol II genes, repair of UV photoproducts in both strands of active and inactive rDNA is inefficient and may result from blockage of """"""""bulky"""""""" excision repair complexes by the nucleolar compartment. We will determine if inefficient repair of active rDNA also occurs for a different """"""""class"""""""" of lesions (N-methyl purines), which are rapidly removed from other genomic sequences by the much smaller base-excision repair proteins. Finally, we are using a simple yeast plasmid, containing an inducible gene and a constitutively expressed gene, as a model chromatin substrate to study transcription-coupled repair in repair proficient (wt) and repair deficient (rad-) yeast cells. We will examine repair at specific sites of this plasmid in """"""""isogenic sets"""""""" of wt/rad-cells to determine if specific RAD genes are required for transcription-coupled repair. Thus, we will examine the effects of gene expression and changes in local chromatin structure on the efficiency of DNA lesions. Since these lesions may alter the expression of specific genes required for establishing the neoplastic phenotype, these studies should provide valuable insight into the cell's defense mechanism for resisting neoplastic transformation by environmental carcinogens.

National Institute of Health (NIH)
National Institute of Environmental Health Sciences (NIEHS)
Research Project (R01)
Project #
Application #
Study Section
Radiation Study Section (RAD)
Project Start
Project End
Budget Start
Budget End
Support Year
Fiscal Year
Total Cost
Indirect Cost
Washington State University
Schools of Arts and Sciences
United States
Zip Code
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
Rodriguez, Yesenia; Hinz, John M; Laughery, Marian F et al. (2016) Site-specific Acetylation of Histone H3 Decreases Polymerase ? Activity on Nucleosome Core Particles in Vitro. J Biol Chem 291:11434-45
Hinz, John M; Mao, Peng; McNeill, Daniel R et al. (2015) Reduced Nuclease Activity of Apurinic/Apyrimidinic Endonuclease (APE1) Variants on Nucleosomes: IDENTIFICATION OF ACCESS RESIDUES. J Biol Chem 290:21067-75
Rodriguez, Yesenia; Hinz, John M; Smerdon, Michael J (2015) Accessing DNA damage in chromatin: Preparing the chromatin landscape for base excision repair. DNA Repair (Amst) 32:113-9
Hinz, John M; Laughery, Marian F; Wyrick, John J (2015) Nucleosomes Inhibit Cas9 Endonuclease Activity in Vitro. Biochemistry 54:7063-6
Meas, Rithy; Mao, Peng (2015) Histone ubiquitylation and its roles in transcription and DNA damage response. DNA Repair (Amst) 36:36-42
Hinz, John M; Czaja, Wioletta (2015) Facilitation of base excision repair by chromatin remodeling. DNA Repair (Amst) 36:91-7
Hinz, John M (2014) Impact of abasic site orientation within nucleosomes on human APE1 endonuclease activity. Mutat Res 766-767:19-24
Hinz, John M (2014) Impact of abasic site orientation within nucleosomes on human APE1 endonuclease activity. Mutat Res 766-767:19-24
Duan, Ming-Rui; Smerdon, Michael J (2014) Histone H3 lysine 14 (H3K14) acetylation facilitates DNA repair in a positioned nucleosome by stabilizing the binding of the chromatin Remodeler RSC (Remodels Structure of Chromatin). J Biol Chem 289:8353-63

Showing the most recent 10 out of 55 publications