RNA polymerase (RNAP) sensitively detects DNA damage as it performs one-dimensional scanning of the template strand, thus initiating transcription-coupled repair (TCR) or triggering apoptosis if the blockage persists. A detailed mechanistic understanding of the causes of transcription blockage and their outcomes should reveal novel modes and targets for selectively killing tumor cells, based upon differences in the genes they express compared to those of normal cells. The formation of long RNA/DNA hybrids (R-loops) during transcription can be a fundamental cause of RNAP arrest, and could produce a number of consequent deleterious effects in cells, including apoptosis. We predict that R-loop formation could be targeted to any transcribed sequence with high efficiency, either by co-transcriptional anchoring of nascent RNA to the DNA template (e.g., by bivalent oligonucleotides which can simultaneously form a triplex with DNA and a duplex with RNA) or by sequestration of the non-template strand by a sequence-specific peptide nucleic acid (PNA), a synthetic DNA mimic with superior DNA binding potential that is resistant to disruption by cellular enzymes. We propose a novel approach to selectively incapacitate a unique cell type with a distinguishable transcription profile, by targeting R-loop formation to a selected active gene. The combination of R-loop formation and arrested RNAP should also strongly block advancing replication forks to stifle proliferation, thus ensuring an even more robust lethal outcome. In fact, the transcription of this particular gene becomes toxic for the cell, regardles of its function and whether or not it is essential. In essence, we intend to make the very act of transcription lethal for the targeted cancer cells without affecting the normal cells. We will pursue the feasibility of this strategy; first, by testing the ability of various PNAs and anchorin oligonucleotides to induce R-loop formation, using in vitro transcription assays with T7 RNAP and RNAP II in HeLa cell nuclear extracts, combined with enzymological approaches and gel electrophoresis to characterize reaction products. Successful in vitro R-loop-inducing reagents will then be introduced into cells and their effects upon transcription, replication and cell lethaity will be monitored. In parallel, we will carry out studies to improve the efficiency of the deliveryof PNA to the target cells. We also propose to extend our studies of intrinsic transcription blockage by G-rich sequences and their biological implications. The results from the proposed studies will provide important insights into the regulation of gene expression and will contribute to the development of novel approaches in chemotherapy for cancer and human genetic disease.

Public Health Relevance

This project will explore a novel approach to achieve selective cell lethality in cancer therapy, in which the very act of transcription becomes toxic, as the nascent RNA product becomes anchored to the DNA template, thereby arresting the RNA polymerase (and blocking subsequent DNA replication) in a gene that is expressed in cells from a tumor but not in normal cells. The studies will also enhance our understanding of the roles of transcription in processing unusual DNA structures that have been implicated in cancer etiology and human hereditary disease.

Agency
National Institute of Health (NIH)
Institute
National Cancer Institute (NCI)
Type
Research Project (R01)
Project #
5R01CA077712-18
Application #
9087126
Study Section
Cancer Etiology Study Section (CE)
Project Start
1998-05-05
Project End
2017-07-31
Budget Start
2016-08-01
Budget End
2017-07-31
Support Year
18
Fiscal Year
2016
Total Cost
Indirect Cost
Name
Stanford University
Department
Biology
Type
Schools of Arts and Sciences
DUNS #
009214214
City
Stanford
State
CA
Country
United States
Zip Code
94304
Pipathsouk, Anne; Belotserkovskii, Boris P; Hanawalt, Philip C (2017) When transcription goes on Holliday: Double Holliday junctions block RNA polymerase II transcription in vitro. Biochim Biophys Acta 1860:282-288
Ganesan, Ann; Hanawalt, Philip (2016) Photobiological Origins of the Field of Genomic Maintenance. Photochem Photobiol 92:52-60
Belotserkovskii, Boris P (2016) Torque-winding interdependence for a flexible polymer chain wound around a cylinder in the presence of obstacles. Phys Rev E 93:032509
Haradhvala, Nicholas J; Polak, Paz; Stojanov, Petar et al. (2016) Mutational Strand Asymmetries in Cancer Genomes Reveal Mechanisms of DNA Damage and Repair. Cell 164:538-49
Spivak, Graciela (2016) Transcription-coupled repair: an update. Arch Toxicol 90:2583-2594
Hanawalt, Philip C (2015) A balanced perspective on unbalanced growth and thymineless death. Front Microbiol 6:504
Hanawalt, Philip C (2015) Historical perspective on the DNA damage response. DNA Repair (Amst) 36:2-7
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
Pandey, Shristi; Ogloblina, Anna M; Belotserkovskii, Boris P et al. (2015) Transcription blockage by stable H-DNA analogs in vitro. Nucleic Acids Res 43:6994-7004
Hanawalt, Phil; Grollman, Arthur; SankarMitra (2015) A tribute in memory of Richard B. (Dick) Setlow (1921-2015). DNA Repair (Amst) 33:111-4

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