title: Transcription blockage by stable H-DNA analogs in vitro.
abstract: DNA sequences that can form unusual secondary structures are implicated in regulating gene expression and causing genomic instability. H-palindromes are an important class of such DNA sequences that can form an intramolecular triplex structure, H-DNA. Within an H-palindrome, the H-DNA and canonical B-DNA are in a dynamic equilibrium that shifts toward H-DNA with increased negative supercoiling. The interplay between H- and B-DNA and the fact that the process of transcription affects supercoiling makes it difficult to elucidate the effects of H-DNA upon transcription. We constructed a stable structural analog of H-DNA that cannot flip into B-DNA, and studied the effects of this structure on transcription by T7 RNA polymerase in vitro. We found multiple transcription blockage sites adjacent to and within sequences engaged in this triplex structure. Triplex-mediated transcription blockage varied significantly with changes in ambient conditions: it was exacerbated in the presence of Mn(2+) or by increased concentrations of K(+) and Li(+). Analysis of the detailed pattern of the blockage suggests that RNA polymerase is sterically hindered by H-DNA and has difficulties in unwinding triplex DNA. The implications of these findings for the biological roles of triple-stranded DNA structures are discussed.
authors.affiliation: Department of Biology, Stanford University, Stanford, CA 94305, USA.
authors.forename: Shristi
authors.initials: S
authors.lastname: Pandey
authors.affiliation: Blokhin Cancer Research Center RAMS, Moscow, Russia.
authors.forename: Anna M
authors.initials: AM
authors.lastname: Ogloblina
authors.affiliation: Department of Biology, Stanford University, Stanford, CA 94305, USA.
authors.forename: Boris P
authors.initials: BP
authors.lastname: Belotserkovskii
authors.affiliation: Department of Chemistry, Lomonosov Moscow State University, Moscow, Russia.
authors.forename: Nina G
authors.initials: NG
authors.lastname: Dolinnaya
authors.affiliation: Blokhin Cancer Research Center RAMS, Moscow, Russia.
authors.forename: Marianna G
authors.initials: MG
authors.lastname: Yakubovskaya
authors.affiliation: Department of Biology, Tufts University, Medford, MA 02155, USA.
authors.forename: Sergei M
authors.initials: SM
authors.lastname: Mirkin
authors.affiliation: Department of Biology, Stanford University, Stanford, CA 94305, USA hanawalt@stanford.edu.
authors.forename: Philip C
authors.initials: PC
authors.lastname: Hanawalt
mesh_terms: D002413:Cations, Divalent; D002414:Cations, Monovalent; D004247:DNA / Q000737:chemistry*; D012321:DNA-Directed RNA Polymerases / Q000378:metabolism; D008670:Metals / Q000737:chemistry; D009690:Nucleic Acid Conformation; D014158:Transcription, Genetic; D014764:Viral Proteins / Q000378:metabolism
chemical_list: D002413:Cations, Divalent; D002414:Cations, Monovalent; D008670:Metals; D014764:Viral Proteins; C051802:triplex DNA; D004247:DNA; C062210:bacteriophage T7 RNA polymerase; D012321:DNA-Directed RNA Polymerases