Nucleotide excision repair (NER) is a highly conserved pathway from bacteria to humans that removes a wide variety of DNA lesions caused by environmental agents such as UV light and air pollutants. In addition, NER is important for the removal of adducts induced by several anticancer drugs, such as cisplatin. One of the fundamental questions in the field of DNA repair is how a modest number of repair proteins scan through several million (for bacteria) to a few billion base-pairs (for mammalian cells) of non-damaged DNA to find rare damaged bases. This project combines single molecule approaches (atomic force microscopy, and oblique angle fluorescence) with biochemical approaches to examine how bacterial and eukaryotic nucleotide excision repair proteins detect and remove damaged nucleotides from DNA. This study uses a novel optical platform for viewing single molecules in real-time moving on DNA and will give a dynamic view of how these protein machines assemble on DNA and track down DNA lesions. This highly innovative project has three main aims: 1) to investigate how bacterial NER proteins achieve highly specific recognition and repair of DNA damage;2) to characterize the search mechanisms employed by human damage recognition proteins, XPC-HR23B, XPA, RPA, and UV-DDB;and 3) to examine the dynamics of human XPD (ERCC2), and XPB (ERCC3) helicase proteins on DNA. This project will test the hypothesis that the bacterial and human NER proteins share similar modes of DNA binding and searching mechanisms for damage detection and processing. Completion of these aims will help to revolutionize the field of DNA repair by developing new imaging techniques that allow direct visualization and real-time measurements of protein complexes in all stages of repair. They will also begin to address how damage is detected in the context of chromatin. In future years, they will also lay the ground work for imaging single-molecules in real time in living cells.

Public Health Relevance

DNA damage recognition by nucleotide excision repair proteins PI: Bennett Van Houten Project Narrative: Nucleotide excision (NER) repair removes damage from our genome that is induced by a wide variety of environmental agents, including UV light and air pollution. Alterations in NER can cause increased mutations, cancer and cell death and manifest in several human disorders including xeroderma pigmentosum, aging and neurodegeneration. This project will gain insight into how these repair proteins detect and remove DNA damage.

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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University of Pittsburgh
Schools of Medicine
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Springall, Luke; Hughes, Craig D; Simons, Michelle et al. (2018) Recruitment of UvrBC complexes to UV-induced damage in the absence of UvrA increases cell survival. Nucleic Acids Res 46:1256-1265
Van Houten, Bennett; Kad, Neil M (2018) Single-cell mutagenic responses and cell death revealed in real time. Proc Natl Acad Sci U S A 115:7168-7170
Beckwitt, Emily C; Kong, Muwen; Van Houten, Bennett (2018) Studying protein-DNA interactions using atomic force microscopy. Semin Cell Dev Biol 73:220-230
Liu, Lili; Kong, Muwen; Gassman, Natalie R et al. (2017) PARP1 changes from three-dimensional DNA damage searching to one-dimensional diffusion after auto-PARylation or in the presence of APE1. Nucleic Acids Res 45:12834-12847
Luo, Ji; Kong, Muwen; Liu, Lili et al. (2017) Optical Control of DNA Helicase Function through Genetic Code Expansion. Chembiochem 18:466-469
Kaufman, Brett A; Van Houten, Bennett (2017) POLB: A new role of DNA polymerase beta in mitochondrial base excision repair. DNA Repair (Amst) 60:A1-A5
Beckwitt, Emily C; Van Houten, Bennett (2017) Molecular cartography of mutational landscapes in melanomas. EMBO J 36:2812-2814
Kong, Muwen; Beckwitt, Emily C; Springall, Luke et al. (2017) Single-Molecule Methods for Nucleotide Excision Repair: Building a System to Watch Repair in Real Time. Methods Enzymol 592:213-257
Kong, Muwen; Van Houten, Bennett (2017) Rad4 recognition-at-a-distance: Physical basis of conformation-specific anomalous diffusion of DNA repair proteins. Prog Biophys Mol Biol 127:93-104
Houten, Bennett Van; Kuper, Jochen; Kisker, Caroline (2016) Role of XPD in cellular functions: To TFIIH and beyond. DNA Repair (Amst) 44:136-142

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