The broad objective of this proposal is to understand the molecular details of DNA excision repair in the context of DNA packaging and gene transcription in chromatin. We will use UV radiation and DNA methylating chemicals as prototype environmental agents for studies on nucleotide excision repair (NER) and base excision repair (BER), respectively. Repair will be examined in a section of a mouse viral gene promoter (GRE) that is packaged in a positioned nucleosome (NCP) and induced upon glucocorticoid hormone receptor (HR) binding in vivo.
In aim I, we will examine the role of NCP unwrapping dynamics in driving complex formation between repair proteins and DNA lesions. These studies will involve the use of restriction enzyme accessibility (REA) and Fvrster resonance energy transfer (FRET) to determine the effect of DNA lesions on NCP unwrapping dynamics. The major form of UV damage in DNA (CTD) and G::U mismatches will be incorporated at specific sites of GRE-containing NCPs and rotationally aligned on the histone surface by bracketing the GRE sequence with NCP positioning elements. Dynamic FRET will be used to monitor the rates of trapping of unwrapped NCPs by DNA repair proteins at site-specific lesions. We will also examine BER of uracil incorporated at specific sites in the GRE complexed with protein (aim II). These studies will involve using purified human enzymes and mammalian cell extracts to determine the effect of nucleosome location, histone modification and HR binding on BER at site-specific uracil bases in the GRE and adjacent sequences. The bracketed GRE sequence containing G::U mismatches will be packaged into nucleosomes containing unmodified or specifically modified histones for comparisons with naked DNA. NER of UV damage (CPDs) and BER of N-methyl purines (NMPs) will also be examined in well- characterized chromatin loci in the yeast S. cerevisiae. We will examine the efficiency of NER of CPDs in histone mutants (sin and Irs) that require less chromatin remodeling during gene activation and whose NCPs are more 'mobile'(aim III). We will focus on removal of CPDs from each strand of active and inactive Pol II genes and ribosomal RNA genes (Pol I) of lrs mutants of repair proficient (wt) and repair deficient (rad) cells. The chromatin structure of these loci is well known and they provide different chromatin 'landscapes'for comparison with wt cells. Finally, we will examine the efficiency of BER of NMPs in sin and lrs mutants of yeast (aim IV). Removal of NMPs from each strand of active and inactive Pol I and Pol II genes of sin and Irs mutants, will be compared between wt and rad mutant cells. Thus, we will use a "multifaceted" approach to examine the role of chromatin structure in DNA repair with the ultimate goal of understanding this process in human cells. Since DNA lesions may alter the expression of specific genes required for establishing the neoplastic phenotype, these studies should also provide valuable insight into the cell's defense mechanism for resisting neoplastic transformation by environmental carcinogens.

Public Health Relevance

DNA damage is detrimental to all living cells and repair of these lesions in mammalian cells is a 'frontline defense'against mutations and cancer. We use prototype DNA-damaging agents to elicit DNA repair in different regions of DNA packaging in cells to determine how cells cope with repair of 'buried'lesions in DNA. Thus, results from our laboratory have implications for the broad spectrum of cancer etiology, prevention and treatment.

National Institute of Health (NIH)
National Institute of Environmental Health Sciences (NIEHS)
Research Project (R01)
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Cancer Etiology Study Section (CE)
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Reinlib, Leslie J
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Washington State University
Schools of Arts and Sciences
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Hinz, John M (2014) Impact of abasic site orientation within nucleosomes on human APE1 endonuclease activity. Mutat Res 766-767:19-24
Rodriguez, Yesenia; Smerdon, Michael J (2013) The structural location of DNA lesions in nucleosome core particles determines accessibility by base excision repair enzymes. J Biol Chem 288:13863-75
Ghosh-Roy, Sounak; Das, Dhiman; Chowdhury, Debarati et al. (2013) Rad26, the transcription-coupled repair factor in yeast, is required for removal of stalled RNA polymerase-II following UV irradiation. PLoS One 8:e72090
Nag, Ronita; Kyriss, McKenna; Smerdon, John W et al. (2010) A cassette of N-terminal amino acids of histone H2B are required for efficient cell survival, DNA repair and Swi/Snf binding in UV irradiated yeast. Nucleic Acids Res 38:1450-60
Hinz, John M; Rodriguez, Yesenia; Smerdon, Michael J (2010) Rotational dynamics of DNA on the nucleosome surface markedly impact accessibility to a DNA repair enzyme. Proc Natl Acad Sci U S A 107:4646-51
Duan, Ming-Rui; Smerdon, Michael J (2010) UV damage in DNA promotes nucleosome unwrapping. J Biol Chem 285:26295-303
Hinz, John M (2010) Role of homologous recombination in DNA interstrand crosslink repair. Environ Mol Mutagen 51:582-603
Hinz, Angela K; Wang, Yan; Smerdon, Michael J (2010) Base excision repair in a glucocorticoid response element: effect of glucocorticoid receptor binding. J Biol Chem 285:28683-90
Nag, Ronita; Smerdon, Michael J (2009) Altering the chromatin landscape for nucleotide excision repair. Mutat Res 682:13-20
Zhang, Ling; Jones, Kristi; Smerdon, Michael J et al. (2009) Assays for chromatin remodeling during nucleotide excision repair in Saccharomyces cerevisiae. Methods 48:19-22

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