Research ArticleSTRUCTURAL BIOLOGY

Structure of transcribed chromatin is a sensor of DNA damage

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Science Advances  03 Jul 2015:
Vol. 1, no. 6, e1500021
DOI: 10.1126/sciadv.1500021
  • Fig. 1 Experimental strategies: Analysis of the effect of NT-SSBs on transcription through chromatin.

    (A) Identification of SSBs inhibiting or facilitating the progression of E. coli RNA polymerase through a nucleosome. Positioned nucleosomes were assembled on end-labeled (at nontemplate strand, asterisk) 603 DNA containing random single SSBs and transcribed for a limited time. Nucleosomes were separated by native PAGE. DNA was purified from transcribed and nontranscribed templates and analyzed by denaturing PAGE. NPS, nucleosome positioning sequence. NT-SSBs facilitating and inhibiting transcription through the nucleosome are shown in blue and red, respectively. (B) Transcription of nucleosomes containing unique SSBs. 603 nucleosomes containing SSBs in position +12, +17, +22, +31, or +50 (nontemplate DNA strand, insert at the bottom) were transcribed for different time intervals in the presence of various concentrations of KCl. Pulse-labeled RNA was separated by denaturing PAGE. The direction of transcription is indicated by the dashed yellow arrow.

  • Fig. 2 Location of intranucleosomal NT-SSBs determines their effect on transcription.

    (A) Transcription of 603 nucleosomes containing end-labeled DNA and random single SSBs by E. coli RNAP for 10 min. Transcribed nucleosomes (N) and nontranscribed elongation complexes containing arrested RNAP (ECs) were separated by native PAGE before or after transcription. DN, nontranscribed dinucleosomes; H, hexasomes. Complexes containing 11-mer RNA are indicated. (B) Analysis of the distribution of NT-SSBs between transcribed (T) and nontranscribed (NT) templates by denaturing PAGE. C, control intact DNA. The nucleosome (blue oval) and the dyad (black rectangle) are indicated. Red and blue lines show DNA regions where breaks in the transcribed fraction were under- or overrepresented, respectively. (C) Quantitative analysis of the distribution of the SSBs shown in (B). M, pBR322 Msp I digest. The overall positive and negative effects of NT-SSBs on transcription (+ and −) roughly correlate with uncoiling of nucleosomal DNA in front and behind the RNAP, respectively (21, 30). Only half of the nucleosomal DNA supercoil is shown.

  • Fig. 3 Individual SSBs differentially affect transcription through a nucleosome.

    (A) Transcription through 603 nucleosomes containing single SSBs at position +12 or +50 by E. coli RNAP for 10 min at 40, 150, 300, or 1000 mM KCl. Analysis of pulse-labeled RNA by denaturing PAGE. (B) A model describing the effect of SSBs on transcription through a nucleosome. As RNAP encounters the histone octamer and proceeds past the SSB, a transient DNA loop could be formed. Formation of the loop would result in accumulation of unconstrained negative (−−) and positive (++) DNA supercoiling behind and in front of the enzyme, respectively. We propose that SSBs facilitate or inhibit transcription through a nucleosome when present in front or behind RNAP, and relieve positive or negative unconstrained DNA supercoiling, respectively. (C) Transcription through 603 nucleosomes containing single SSBs by E. coli RNAP for 0.5, 1, and 10 min at 300 mM KCl. Analysis of pulse-labeled RNA by denaturing PAGE. (D) Quantitative analysis of run-off transcripts shown in (C). The quantified signals were normalized to the corresponding 10-min signals. The signals without normalization are shown in fig. S2.

  • Fig. 4 +12 NT-SSB relieves nucleosomal pausing and induces a strong arrest of Pol II in a nucleosome.

    (A) The experimental approach for analysis of transcription through 603 nucleosome by yeast Pol II. Pol II elongation complex (EC) was assembled, immobilized on Ni2+-NTA agarose beads, and ligated to DNA or nucleosomal templates. Pol II was advanced to produce EC-5 complex using a subset of NTPs and [α-32P]GTP (guanosine triphosphate) to label the RNA. Then transcription was resumed by the addition of all unlabeled NTPs. (B) Transcription through 603 nucleosomes containing single SSB at position +12 by Pol II for 10 min at 40, 150, 300, or 1000 mM KCl. Analysis of pulse-labeled RNA by denaturing PAGE.

  • Fig. 5 NT-SSBs induce a strong arrest of RNAP in discrete 10-bp periodic positions of nucleosomal DNA.

    (A) Predictions of the looping model (Fig. 3B) for the effect of NT-SSBs on the arrest of RNAP during transcription through chromatin. Different sets of closely located SSBs (larger arrows) are expected to induce arrest of RNAP at different, distinct locations on nucleosomal DNA (see Results for detail). (B) Transcription through 603 nucleosomes containing single SSBs at position +12, +17, +22, or +31 by E. coli RNAP for 2 min at 150 or 300 mM KCl. Analysis of pulse-labeled RNA by denaturing PAGE. (C) Quantitative analysis of SSB-induced arrest of RNAP in the nucleosome.

  • Fig. 6 Proposed mechanism of NT-SSB–induced arrest of Pol II in chromatin.

    Only half of the nucleosomal DNA supercoil is shown. As Pol II encounters the histone octamer, its progression is hindered, and positive unconstrained DNA supercoiling (++) accumulates in front of the enzyme. We propose that single-strand breaks in DNA facilitate transcription through a nucleosome when present in front of the enzyme (perhaps by relieving unconstrained positive DNA supercoiling) (complex 1). Then, nucleosomal DNA is partially uncoiled from the octamer (complex 2), and transient DNA loops are formed [likely at the position of the active center of RNA polymerase +24, +34, or +44 (complex 3)]. Here, SSBs could inhibit transcription and DNA displacement (steps 3 to 4) when present behind RNAP, perhaps by relieving negative supercoiling behind the enzyme. Once Pol II proceeds to position +49, a very small intranucleosomal DNA loop is formed (complex 5), DNA is uncoiled from the octamer in front of the enzyme, and transcription continues efficiently (21).

Supplementary Materials

  • Supplementary material for this article is available at http://advances.sciencemag.org/cgi/content/full/1/6/e1500021/DC1

    Discussion

    Fig. S1. Effect of NT-SSBs on transcription of histone-free DNA.

    Fig. S2. Quantitative analysis of run-off transcripts shown in Fig. 3C without normalization to the corresponding 10-min signals.

    Fig. S3. Encounter between RNAP and nucleosome likely results in formation of topological locks.

    Reference (50)

  • Supplementary Materials

    This PDF file includes:

    • Discussion
    • Fig. S1. Effect of NT-SSBs on transcription of histone-free DNA.
    • Fig. S2. Quantitative analysis of run-off transcripts shown in Fig. 3C without normalization to the corresponding 10-min signals.
    • Fig. S3. Encounter between RNAP and nucleosome likely results in formation of topological locks.
    • Reference (50)

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