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.
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.
|Ezerskyte, Monika; Paredes, João A; Malvezzi, Stefano et al. (2018) O6-methylguanine-induced transcriptional mutagenesis reduces p53 tumor-suppressor function. Proc Natl Acad Sci U S A 115:4731-4736|
|Kolbanovskiy, Marina; Chowdhury, Moinuddin A; Nadkarni, Aditi et al. (2017) The Nonbulky DNA Lesions Spiroiminodihydantoin and 5-Guanidinohydantoin Significantly Block Human RNA Polymerase II Elongation in Vitro. Biochemistry 56:3008-3018|
|Nadkarni, Aditi; Burns, John A; Gandolfi, Alberto et al. (2016) Nucleotide Excision Repair and Transcription-coupled DNA Repair Abrogate the Impact of DNA Damage on Transcription. J Biol Chem 291:848-61|
|Dreij, Kristian; Rhrissorrakrai, Kahn; Gunsalus, Kristin C et al. (2010) Benzo[a]pyrene diol epoxide stimulates an inflammatory response in normal human lung fibroblasts through a p53 and JNK mediated pathway. Carcinogenesis 31:1149-57|
|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|
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