Role of transcription in genomic stability RNA polymerase (RNAP) sensitively detects damage as it performs one-dimensional scanning of template DNA, thus initiating the dedicated pathway of transcription-coupled repair (TCR). However, transcription can also increase mutagenesis. Hot spots for genomic instability implicated in human genetic disease and carcinogenesis have been localized in DNA sequences that can adopt non- canonical DNA structures (e.g. Z-DNA, H-DNA, G4-DNA, triplet repeats and palindromes forming hairpins/cruciforms). We wish to gain a mechanistic perspective on possible outcomes when RNAP encounters such structures. We hypothesize that TCR may be mutagenic when it occurs at or near non-canonical DNA structures, certain lesion types, or bound complex ligands. To test this hypothesis we will determine the precise signals that can arrest RNAP and elicit TCR, to learn whether TCR might be error-prone under some circumstances. An in vitro transcription assay using purified T7RNAP and mammalian RNAPII with required factors on defined DNA substrates will be utilized to: (1) Characterize RNAP arrested at site-specific non-canonical DNA structures and lesions, including psoralen monoadducts vs. interstrand crosslinks, and adducts of the acylfulvenes, which are reportedly subject to TCR but not global excision repair. The arrested RNAP, transcription bubble, and RNA/DNA hybrid will be mapped. Effects of added factors such as TFIIS, CSB and TFIIH, which may modulate transcription arrest, as well as effects of mismatch repair proteins and RecQ, which modulate some non B-form DNA structures will be determined. Recognition of non-canonical structures by repair enzymes in human cell extracts will be assessed (2) Evaluate cooperative effects on transcription of abasic sites or 8oxoGuanine introduced into Z-DNA, and effects of complex ligands, such as the Z-DNA binding protein ADAR1, and topoisomerase 1 trapped at abasic sites. (3) Utilize stable complexes of peptide nucleic acid (PNA) to explore novel aspects of these unique ligands for targeted gene alterations, while revealing mechanistic details of transcriptional processing. PNA binding will also be explored as an alternative to promoter-driven transcription, to possibly achieve higher efficiency of substrate usage, toward improved assays for transcription behavior at lesions and cell-free TCR assays.
The results from this project will enhance our understanding of the roles of transcription and TCR in processing lesions and other abnormalities in DNA that have been implicated in human disease. Since prolonged transcription arrest generates a strong signal for apoptosis, the research may lead to novel modes of chemotherapy, involving selective inhibition of TCR in target cells combined with administration of transcription-blocking drugs.
|Belotserkovskii, Boris P; Hanawalt, Philip C (2015) PNA binding to the non-template DNA strand interferes with transcription, suggesting a blockage mechanism mediated by R-loop formation. Mol Carcinog 54:1508-12|
|Belotserkovskii, Boris P; Neil, Alexander J; Saleh, Syed Shayon et al. (2013) Transcription blockage by homopurine DNA sequences: role of sequence composition and single-strand breaks. Nucleic Acids Res 41:1817-28|
|Belotserkovskii, Boris P; Hanawalt, Philip C (2011) Anchoring nascent RNA to the DNA template could interfere with transcription. Biophys J 100:675-84|
|Belotserkovskii, Boris P; Liu, Richard; Tornaletti, Silvia et al. (2010) Mechanisms and implications of transcription blockage by guanine-rich DNA sequences. Proc Natl Acad Sci U S A 107:12816-21|
|Belotserkovskii, Boris P; Liu, Richard; Hanawalt, Philip C (2009) Peptide nucleic acid (PNA) binding and its effect on in vitro transcription in friedreich's ataxia triplet repeats. Mol Carcinog 48:299-308|
|Hanawalt, Philip C (2008) Emerging links between premature ageing and defective DNA repair. Mech Ageing Dev 129:503-5|
|Belotserkovskii, Boris P; De Silva, Erandi; Tornaletti, Silvia et al. (2007) A triplex-forming sequence from the human c-MYC promoter interferes with DNA transcription. J Biol Chem 282:32433-41|
|Hsu, Pei-hsin; Hanawalt, Philip C; Nouspikel, Thierry (2007) Nucleotide excision repair phenotype of human acute myeloid leukemia cell lines at various stages of differentiation. Mutat Res 614:3-15|
|Cline, Susan D; Hanawalt, Philip C (2006) Topoisomerase deficiencies subtly enhance global genomic repair of ultraviolet-induced DNA damage in Saccharomyces cerevisiae. DNA Repair (Amst) 5:611-7|
|Nouspikel, Thierry; Hanawalt, Philip C (2006) Impaired nucleotide excision repair upon macrophage differentiation is corrected by E1 ubiquitin-activating enzyme. Proc Natl Acad Sci U S A 103:16188-93|
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