The broad objective of this proposal is to understand the molecular details of DNA repair in the context of DNA packaging and gene transcription in chromatin. The repair proteins represent 'molecular guardians' of genomic integrity and appear to be one of the first lines of defense aqainst certain human diseases, including both spontaneous and environmentally induced cancers. DNA damage results from a wide variety of environmental agents, such as ultraviolet (UV) radiation and chemical carcinogens, giving rise to heritable changes in the sequence of genes. UV radiation and dimethylsulfate will be used as prototype environmental agents for studies on nucleotide excision repair (NER) and base excision repair (BER), respectively. We will examine the relationship between DNA repair, transcription and chromatin structure of different classes of genes in well-defined protein complexes in vitro, as well as in intact yeast cells. Repair will be examined in a section of a mouse viral gene promoter (LTR), that is packaged in a positioned nucleosome and induced upon binding of a glucocorticoid hormone receptor (HR) in vivo. The major form of UV damage in DNA (CTD) has been synthesized and will be incorporated into specific sites of the LTR for studies on the effect of CTD damage on HR binding. This sequence will also be bracketed by nucleosome positioning elements and packaged into a nucleosome for studies on the effect of CTD location and orientation in a nucleosome on NER efficiency in Xenopus extracts. G::U mismatches will also be synthesized into the LTR sequence to examine the effect of HR bindin,q and nucleosome formation on BER efficiency, using purified human BER proteins and mammalian cell extracts. NER of UV damaqe (CPDs) and BER of N-methyl purines (NMPs) will also be examined in well-characterized, inducible yeast RNA pol II genes (Gal1-10 and PH05), and active and inactive ribosomal RNA pol I genes (rDNA). The chromatin structure of these different gene loci has been exquisitely mapped in both the inactive and active gene states. We will examine the efficiency of global genome repair (GGR) and transcription coupled repair (TCR) of CPDs, as well as BER of NMPs, in histone gene mutants (sin and Irs) that show less need for chromatin remodeling during gene activation and whose nucleosomes are more 'mobile' on DNA in vitro. Therefore, NER (GGR or TCR) and/or BER may function more efficiently in regions of positioned nucleosomes in these mutants, providinq a link between DNA repair and chromatin remodelin,q in intact cells. Thus, we will examine the effects of gene expression and changes in local chromatin structure on the efficiency of DNA repair. Since these lesions may alter the expression of specific genes required for establishing the neoplastic phlenotype, these studies should provide insiqht into the cell's defense mechanism for resisting transformation by environmental carcinogens.

National Institute of Health (NIH)
National Institute of Environmental Health Sciences (NIEHS)
Research Project (R01)
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Special Emphasis Panel (ZRG1-CDF-2 (90))
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Reinlib, Leslie J
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Washington State University
Schools of Arts and Sciences
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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; 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; 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 (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

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