DNA damage-dependent transcriptional arrest and termination of RNA polymerase II elongation complexes in DNA template containing HIV-1 promoter.
We have developed a new biochemical method to isolate a homogeneous population of RNA polymerase II (RNA pol II) elongation complexes arrested at a DNA damage site. The method involves triple-helix formation at a predetermined site in DNA template with a third strand labeled with psoralen at its 5'-end and a biotin at the 3'-end. After triplex formation and near-ultraviolet irradiation (360 nm), DNA templates modified with psoralen were immobilized on streptavidin-coated magnetic beads and used for in vitro transcription reactions with HeLa nuclear extracts. Separation of magnetic beads from solution results in isolation of arrested elongation complexes on the immobilized DNA templates. We have applied the method to arrest RNA pol II elongation complexes on a DNA template containing HIV-1 promoter. Our results indicate that psoralen crosslink in the template strand efficiently arrests elongation complexes, and psoralen monoadducts terminate transcription. Our results also demonstrate that a triple-helical structure stabilized by an intercalator, acridine, attached to the third strand of the helix inhibits transcription by a termination pathway. Isolation of stable RNA pol II elongation complexes arrested at DNA damage sites is a remarkable finding. This result demonstrates that arrested elongation complexes are impervious to DNA damage repair machinery and other regulatory proteins present in HeLa nuclear extracts. The method of delivering site-specific psoralen damage by a triplex structure and isolation of arrested RNA pol II elongation complexes should be generalizable to any promoter and DNA template sequences. This strategy provides a new approach to study the mechanism of transcription elongation and transcription-coupled DNA damage repair.
Wang
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Department of Pharmacology, Robert Wood Johnson Medical School, University of Medicine and Dentistry of New Jersey, Piscataway, NJ 08854, USA.
Rana
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D001483:Base Sequence; D004247:DNA / Q000235:genetics; D004249:DNA Damage; D004260:DNA Repair; D015497:HIV-1 / Q000235:genetics*; D006367:HeLa Cells; D006801:Humans; D008969:Molecular Sequence Data; D010445:Peptide Elongation Factors / Q000235:genetics; D011401:Promoter Regions, Genetic / Q000235:genetics*; D012319:RNA Polymerase II / Q000235:genetics* / Q000378:metabolism; D014158:Transcription, Genetic
D010445:Peptide Elongation Factors; D004247:DNA; D012319:RNA Polymerase II