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.

Agency
National Institute of Health (NIH)
Institute
National Institute of Environmental Health Sciences (NIEHS)
Type
Research Project (R01)
Project #
5R01ES019566-04
Application #
8580937
Study Section
Cancer Etiology Study Section (CE)
Program Officer
Reinlib, Leslie J
Project Start
2010-12-09
Project End
2015-11-30
Budget Start
2013-12-01
Budget End
2014-11-30
Support Year
4
Fiscal Year
2014
Total Cost
$362,954
Indirect Cost
$118,444
Name
University of Pittsburgh
Department
Pharmacology
Type
Schools of Medicine
DUNS #
004514360
City
Pittsburgh
State
PA
Country
United States
Zip Code
15213
Machado, Carlos R; Vieira-da-Rocha, João P; Mendes, Isabela Cecilia et al. (2014) Nucleotide excision repair in Trypanosoma brucei: specialization of transcription-coupled repair due to multigenic transcription. Mol Microbiol 92:756-76
Van Houten, Bennett; Kad, Neil (2014) Investigation of bacterial nucleotide excision repair using single-molecule techniques. DNA Repair (Amst) 20:41-8
Ghodke, Harshad; Wang, Hong; Hsieh, Ching L et al. (2014) Single-molecule analysis reveals human UV-damaged DNA-binding protein (UV-DDB) dimerizes on DNA via multiple kinetic intermediates. Proc Natl Acad Sci U S A 111:E1862-71
Hughes, Craig D; Simons, Michelle; Mackenzie, Cassidy E et al. (2014) Single molecule techniques in DNA repair: a primer. DNA Repair (Amst) 20:2-13
Kad, Neil M; Van Houten, Bennett (2014) Single molecule approaches: watching DNA repair one molecule at a time. Preface. DNA Repair (Amst) 20:1
Van Houten, Bennett; Kisker, Caroline (2014) Transcriptional pausing to scout ahead for DNA damage. Proc Natl Acad Sci U S A 111:3905-6
Lan, Li; Nakajima, Satoshi; Wei, Leizhen et al. (2014) Novel method for site-specific induction of oxidative DNA damage reveals differences in recruitment of repair proteins to heterochromatin and euchromatin. Nucleic Acids Res 42:2330-45
Pullara, Filippo; Guerrero-Santoro, Jennifer; Calero, Monica et al. (2013) A general path for large-scale solubilization of cellular proteins: from membrane receptors to multiprotein complexes. Protein Expr Purif 87:111-9
Hughes, Craig D; Wang, Hong; Ghodke, Harshad et al. (2013) Real-time single-molecule imaging reveals a direct interaction between UvrC and UvrB on DNA tightropes. Nucleic Acids Res 41:4901-12
Vaisman, Alexandra; McDonald, John P; Huston, Donald et al. (2013) Removal of misincorporated ribonucleotides from prokaryotic genomes: an unexpected role for nucleotide excision repair. PLoS Genet 9:e1003878

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