This highly innovative project consists of four aims and investigates the dynamics of eukaryotic nucleotide excision repair proteins at the single molecule level. Specifically, this study analyzes DNA damage through five discrete steps involving: i) initial non-target DNA binding by a NER recognition complex; ii) diffusion of this repair complex to a lesion site; iii) lesion processing, conformational proof reading by this complex (3) (such as insertion of a beta-hairpin into the DNA (Rad4/XPC); iv) arrival of a DNA repair recognition complex ; and v) lesion processing by the second repair complex causing the first repair complex to diffuse away from the damaged site. We hypothesize that the efficiency of the hand-off from one protein complex to the next is the rate-limiting step(s) in NER.
Aim 1 investigates the interactions of purified Rad4-Rad23-Rad33 or XPC- RAD23B-CETN2 complexes with damaged DNA.
The second aim measures the kinetic interactions of Rad14 or XPA with damaged DNA. The particularly original third aim studies the interaction of specific NER damage recognition components in whole-cell extracts of Saccharomyces cerevisiae. Genetic ablation or site-directed mutation into specific genetic loci will allow analysis of the important domains that are essential for damage recognition and lesion hand-off.
The fourth aim measures the damage handoff from UV-DDB to XPC-RAD23B and XPA and how ubiquitylation or PARylation stimulates this process. This project will give an unprecedented view of the complex process of damage recognition steps of eukaryotic nucleotide excision repair and answer several key questions regarding damage recognition that have been intractable in the absence of single molecule approaches. Completion of this project will have a long and lasting impact on the field.

Public Health Relevance

This highly innovative project is focused on understanding the mechanisms by which human DNA repair proteins find and process DNA damage embedded in long-stretches of non- damaged DNA. Loss of DNA repair processes results in accumulation of environmental damage to our genetic material that can manifest as mutations in critical genes enabling oncogenic transformation.

Agency
National Institute of Health (NIH)
Institute
National Institute of Environmental Health Sciences (NIEHS)
Type
Research Project (R01)
Project #
5R01ES019566-07
Application #
9199084
Study Section
Special Emphasis Panel (ZRG1)
Program Officer
Reinlib, Leslie J
Project Start
2010-12-09
Project End
2020-11-30
Budget Start
2016-12-01
Budget End
2017-11-30
Support Year
7
Fiscal Year
2017
Total Cost
Indirect Cost
Name
University of Pittsburgh
Department
Pharmacology
Type
Schools of Medicine
DUNS #
004514360
City
Pittsburgh
State
PA
Country
United States
Zip Code
15213
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
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
Luo, Ji; Kong, Muwen; Liu, Lili et al. (2017) Optical Control of DNA Helicase Function through Genetic Code Expansion. Chembiochem 18:466-469
Beckwitt, Emily C; Van Houten, Bennett (2017) Molecular cartography of mutational landscapes in melanomas. EMBO J 36:2812-2814
Houten, Bennett Van; Kuper, Jochen; Kisker, Caroline (2016) Role of XPD in cellular functions: To TFIIH and beyond. DNA Repair (Amst) 44:136-142
Kad, Neil M; Van Houten, Bennett (2016) DNA repair: Clamping down on copy errors. Nature 539:498-499
Van Houten, Bennett (2016) A tale of two cities: A tribute to Aziz Sancar's Nobel Prize in Chemistry for his molecular characterization of NER. DNA Repair (Amst) 37:A3-A13
He, Jianjun; Wang, Yi; Missinato, Maria A et al. (2016) A genetically targetable near-infrared photosensitizer. Nat Methods 13:263-8
Kong, Muwen; Liu, Lili; Chen, Xuejing et al. (2016) Single-Molecule Imaging Reveals that Rad4 Employs a Dynamic DNA Damage Recognition Process. Mol Cell 64:376-387

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