The long range goal of this research is to gain a detailed understanding of how covalently modified bases in DNA affect RNA polymerase behavior during transcription, and to assess the subsequent cellular responses at the level of DNA repair and transcript integrity. RNA polymerases act as sensors of DNA damage when they stall at lesions in the genome, sometimes triggering damage clearance via transcription-coupled DNA repair, which overlaps with nucleotide excision repair and requires at least two additional proteins that are defective in the disease Cockayne syndrome. But the overlap of TCR with other DNA repair pathways such as base excision repair has not been unequivocally demonstrated or disproved. In contrast to DNA damage that stalls transcription complex progression, some lesions in DNA permit partial or complete transcriptional bypass, resulting in the production of full-length RNA that can contain base misinsertions or deletions, potentially compromising the nascent transcript's function via "transcriptional mutagenesis." Such changes to mRNA can result in altered proteins that affect cell physiology in fundamental ways, possibly triggering disease. Hence, the health-related problems associated with compromised transcription past DNA damage in expressed genes are potentially severe, and yet our basic understanding in this area in human cells is quite limited. In this application we propose experiments to examine the effect of DNA damage on transcription and to decipher further the mechanism of transcription coupled DNA repair. This work will be done in human cells, taking the work beyond the biochemical approaches used thus far. There are three specific aims to address these goals. We will: (1) investigate RNA polymerase II transcription past select DNA adducts;(2) determine the base sequence of the mRNA produced via bypass of each lesion;and (3) examine DNA repair, including TCR, in the site-specifically modified vector. Computer-modeling studies will play role in the continued interpretation of our results by providing molecular models of RNA polymerase II when it encounters a DNA adduct. This research shifts the long-standing emphasis from the effects of DNA lesions on DNA replication, which is important in cells undergoing growth and division, to the role DNA damage plays in RNA synthesis, a process that occurs in all cells, including those that are undergoing division or are terminally differentiated. This research will increase our understanding of the deleterious effect that environmental genotoxic agents have on transcription in humans. While such agents are often associated with mutations and cancer, they may well pose threats to non-dividing cells and disturb RNA synthesis during growth and development, adding to their impact on human health.

Public Health Relevance

Endogenous and exogenous chemicals damage DNA, compromising its ability to store information and transmit it within cells. This research studies how cells repair this damage, preserving DNA and permitting genes to function properly. The studies will improve our understanding of cancer and developmental diseases.

Agency
National Institute of Health (NIH)
Institute
National Institute of Environmental Health Sciences (NIEHS)
Type
Research Project (R01)
Project #
5R01ES010581-10
Application #
8196721
Study Section
Special Emphasis Panel (ZRG1-OBT-P (02))
Program Officer
Shaughnessy, Daniel
Project Start
2000-09-30
Project End
2013-10-31
Budget Start
2011-11-01
Budget End
2012-10-31
Support Year
10
Fiscal Year
2012
Total Cost
$301,466
Indirect Cost
$100,991
Name
New York University
Department
Biology
Type
Schools of Arts and Sciences
DUNS #
041968306
City
New York
State
NY
Country
United States
Zip Code
10012
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Burns, John A; Dreij, Kristian; Cartularo, Laura et al. (2010) O6-methylguanine induces altered proteins at the level of transcription in human cells. Nucleic Acids Res 38:8178-87
Dimitri, Alexandra; Burns, John A; Broyde, Suse et al. (2008) Transcription elongation past O6-methylguanine by human RNA polymerase II and bacteriophage T7 RNA polymerase. Nucleic Acids Res 36:6459-71
Dimitri, Alexandra; Goodenough, Angela K; Guengerich, F Peter et al. (2008) Transcription processing at 1,N2-ethenoguanine by human RNA polymerase II and bacteriophage T7 RNA polymerase. J Mol Biol 375:353-66
Dimitri, Alexandra; Jia, Lei; Shafirovich, Vladimir et al. (2008) Transcription of DNA containing the 5-guanidino-4-nitroimidazole lesion by human RNA polymerase II and bacteriophage T7 RNA polymerase. DNA Repair (Amst) 7:1276-88
Perlow-Poehnelt, Rebecca A; Likhterov, Ilya; Wang, Lihua et al. (2007) Increased flexibility enhances misincorporation: temperature effects on nucleotide incorporation opposite a bulky carcinogen-DNA adduct by a Y-family DNA polymerase. J Biol Chem 282:1397-408
Scicchitano, David A (2005) Transcription past DNA adducts derived from polycyclic aromatic hydrocarbons. Mutat Res 577:146-54
Perlow-Poehnelt, Rebecca A; Likhterov, Ilya; Scicchitano, David A et al. (2004) The spacious active site of a Y-family DNA polymerase facilitates promiscuous nucleotide incorporation opposite a bulky carcinogen-DNA adduct: elucidating the structure-function relationship through experimental and computational approaches. J Biol Chem 279:36951-61
Scicchitano, David A; Olesnicky, Eugenia C; Dimitri, Alexandra (2004) Transcription and DNA adducts: what happens when the message gets cut off? DNA Repair (Amst) 3:1537-48
Schinecker, Thomas M; Perlow, Rebecca A; Broyde, Suse et al. (2003) Human RNA polymerase II is partially blocked by DNA adducts derived from tumorigenic benzo[c]phenanthrene diol epoxides: relating biological consequences to conformational preferences. Nucleic Acids Res 31:6004-15

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