The dynamic response of upstream DNA to transcription-generated torsional stress

The torsional stress caused by counter-rotation of the transcription machinery and template generates supercoils in a closed topological domain, but has been presumed to be too short-lived to be significant in an open domain. This report shows that transcribing RNA polymerases dynamically sustain sufficient torsion to perturb DNA structure even on linear templates. Assays to capture and measure transcriptionally generated torque and to trap short-lived perturbations in DNA structure and conformation showed that the transient forces upstream of active promoters are large enough to drive the supercoil-sensitive far upstream element (FUSE) of the human c-myc into single-stranded DNA. An alternative non-B conformation of FUSE found in stably supercoiled DNA is not accessible dynamically. These results demonstrate that dynamic disturbance of DNA structure provides a real-time measure of ongoing genetic activity.

[1]  L. Rothman-Denes,et al.  Supercoil-induced extrusion of a regulatory DNA hairpin. , 1997, Proceedings of the National Academy of Sciences of the United States of America.

[2]  D. Levens,et al.  A sequence-specific, single-strand binding protein activates the far upstream element of c-myc and defines a new DNA-binding motif. , 1994, Genes & development.

[3]  J. Wang,et al.  Template supercoiling by a chimera of yeast GAL4 protein and phage T7 RNA polymerase. , 1990, Science.

[4]  GC-rich flanking tracts decrease the kinetics of intramolecular DNA triplex formation. , 1992, The Journal of biological chemistry.

[5]  A. Podtelezhnikov,et al.  Large-scale effects of transcriptional DNA supercoiling in vivo. , 1999, Journal of molecular biology.

[6]  R. Wells,et al.  The facile generation of covalently closed, circular DNAs with defined negative superhelical densities. , 1982, Analytical biochemistry.

[7]  John F. Marko,et al.  DNA UNDER HIGH TENSION : OVERSTRETCHING, UNDERTWISTING, AND RELAXATION DYNAMICS , 1998 .

[8]  David Levens,et al.  The FBP interacting repressor targets TFIIH to inhibit activated transcription. , 2000, Molecular cell.

[9]  D. Lilley,et al.  Generation of Superhelical Torsion by ATP-Dependent Chromatin Remodeling Activities , 2000, Cell.

[10]  David Levens,et al.  Transcriptional Consequences of Topoisomerase Inhibition , 2001, Molecular and Cellular Biology.

[11]  R. Wells,et al.  Intramolecular DNA triplexes in supercoiled plasmids. , 1988, Proceedings of the National Academy of Sciences of the United States of America.

[12]  J. Wang,et al.  Supercoiling of the DNA template during transcription. , 1987, Proceedings of the National Academy of Sciences of the United States of America.

[13]  R. Sinden.  DNA Structure and Function , 1994 .

[14]  D. Levens,et al.  Loss of FBP function arrests cellular proliferation and extinguishes c‐myc expression , 2000, The EMBO journal.

[15]  R. Hoess,et al.  Studies on the properties of P1 site-specific recombination: Evidence for topologically unlinked products following recombination , 1983, Cell.

[16]  D. Reinberg,et al.  Defective Interplay of Activators and Repressors with TFIIH in Xeroderma Pigmentosum , 2001, Cell.

[17]  R. Wells,et al.  Intramolecular DNA triplexes in supercoiled plasmids. II. Effect of base composition and noncentral interruptions on formation and stability. , 1989, The Journal of biological chemistry.

[18]  Vitaly Epshtein,et al.  Transcription through the roadblocks: the role of RNA polymerase cooperation , 2003, The EMBO journal.

[19]  C. Benham,et al.  Sites of predicted stress-induced DNA duplex destabilization occur preferentially at regulatory loci. , 1993, Proceedings of the National Academy of Sciences of the United States of America.

[20]  Hiroyasu Itoh,et al.  Direct observation of DNA rotation during transcription by Escherichia coli RNA polymerase , 2001, Nature.

[21]  P. Dröge,et al.  Transcription-driven site-specific DNA recombination in vitro. , 1993, Proceedings of the National Academy of Sciences of the United States of America.

[22]  K. Matsumoto,et al.  Induction of DNA replication by transcription in the region upstream of the human c-myc gene in a model replication system , 1996, Molecular and cellular biology.

[23]  F. Leng,et al.  Potent stimulation of transcription-coupled DNA supercoiling by sequence-specific DNA-binding proteins , 2002, Proceedings of the National Academy of Sciences of the United States of America.

[24]  D. Natale,et al.  Stable DNA unwinding, not "breathing," accounts for single-strand-specific nuclease hypersensitivity of specific A+T-rich sequences. , 1988, Proceedings of the National Academy of Sciences of the United States of America.

[25]  Richard R. Sinden,et al.  DNA structural transitions within the PKD1 gene , 1999, Nucleic Acids Res..

[26]  A. Klug,et al.  Helical periodicity of DNA determined by enzyme digestion , 1980, Nature.

[27]  G W Hatfield,et al.  Transcriptional coupling between the divergent promoters of a prototypic LysR-type regulatory system, the ilvYC operon of Escherichia coli. , 1999, Proceedings of the National Academy of Sciences of the United States of America.

[28]  L. J. Peck,et al.  Energetics of B-to-Z transition in DNA. , 1983, Proceedings of the National Academy of Sciences of the United States of America.

[29]  D. Eick,et al.  Multiple single-stranded cis elements are associated with activated chromatin of the human c-myc gene in vivo , 1996, Molecular and cellular biology.

[30]  T. Tomonaga,et al.  Unrestraining genetic processes with a protein-DNA hinge. , 1998, Molecular cell.

[31]  J. Gralla,et al.  KMnO4 as a probe for lac promoter DNA melting and mechanism in vivo. , 1989, The Journal of biological chemistry.

[32]  Nicholas R. Cozzarelli,et al.  DNA topology and its biological effects , 1990 .

[33]  Z. Wang,et al.  Long-range effects in a supercoiled DNA domain generated by transcription in vitro. , 1997, Journal of molecular biology.

[34]  L. Rothman-Denes,et al.  Sequence-dependent extrusion of a small DNA hairpin at the N4 virion RNA polymerase promoters. , 1998, Journal of molecular biology.

[35]  Daiya Takai,et al.  Origins of bidirectional promoters: computational analyses of intergenic distance in the human genome. , 2003, Molecular biology and evolution.

[36]  W. Keller.  Determination of the number of superhelical turns in simian virus 40 DNA by gel electrophoresis. , 1975, Proceedings of the National Academy of Sciences of the United States of America.

[37]  Jan Greve,et al.  Unfolding individual nucleosomes by stretching single chromatin fibers with optical tweezers , 2001, Nature Structural Biology.

[38]  K. V. van Holde,et al.  Unusual DNA structures, chromatin and transcription. , 1994, BioEssays : news and reviews in molecular, cellular and developmental biology.

[39]  R. Hoess,et al.  Linking-number changes in the DNA substrate during Cre-mediated loxP site-specific recombination. , 1986, Journal of molecular biology.

[40]  S. Cawley,et al.  Unbiased Mapping of Transcription Factor Binding Sites along Human Chromosomes 21 and 22 Points to Widespread Regulation of Noncoding RNAs , 2004, Cell.

[41]  P. Nelson.  Transport of torsional stress in DNA. , 1999, Proceedings of the National Academy of Sciences of the United States of America.

[42]  R. Sinden,et al.  Length-dependent structure formation in Friedreich ataxia (GAA)n*(TTC)n repeats at neutral pH. , 2004, Nucleic acids research.

[43]  M. Lieber,et al.  Bidirectional Gene Organization A Common Architectural Feature of the Human Genome , 2002, Cell.

[44]  Tetsuo Ashizawa,et al.  Unpaired structures in SCA10 (ATTCT)n.(AGAAT)n repeats. , 2003, Journal of molecular biology.

[45]  Michelle D. Wang,et al.  Mechanical disruption of individual nucleosomes reveals a reversible multistage release of DNA , 2002, Proceedings of the National Academy of Sciences of the United States of America.

[46]  D. Kowalski,et al.  Altered DNA conformations detected by mung bean nuclease occur in promoter and terminator regions of supercoiled pBR322 DNA. , 1985, Nucleic acids research.