Transcription blockage by homopurine DNA sequences: role of sequence composition and single-strand breaks

Abstract

The ability of DNA to adopt non-canonical structures can affect transcription and has broad implications for genome functioning. We have recently reported that guanine-rich (G-rich) homopurine-homopyrimidine sequences cause significant blockage of transcription in vitro in a strictly orientation-dependent manner: when the G-rich strand serves as the non-template strand [Belotserkovskii et al. (2010) Mechanisms and implications of transcription blockage by guanine-rich DNA sequences., Proc. Natl Acad. Sci. USA, 107, 12816-12821]. We have now systematically studied the effect of the sequence composition and single-stranded breaks on this blockage. Although substitution of guanine by any other base reduced the blockage, cytosine and thymine reduced the blockage more significantly than adenine substitutions, affirming the importance of both G-richness and the homopurine-homopyrimidine character of the sequence for this effect. A single-strand break in the non-template strand adjacent to the G-rich stretch dramatically increased the blockage. Breaks in the non-template strand result in much weaker blockage signals extending downstream from the break even in the absence of the G-rich stretch. Our combined data support the notion that transcription blockage at homopurine-homopyrimidine sequences is caused by R-loop formation.

Figure 1.

Experimental strategy. DNA strands are shown as thin black lines, except within a specific insert (e.g. homopurine-homopyrimidine sequence) where they are shown by thick gray lines; RNA strand is shown as a thick black line; RNA polymerase (RNAP) is shown as a gray oval with dotted borders. The distance from the transcription start site to the insert is ∼0.25 kb; the sites for nicking enzymes are localized 2–9 nt from the insert. In most experiments, HindIII-digested templates were used, which produce run-off ∼0.5 kb.

Figure 2.

Transcription blockage for various DNA insert sequences. The sequences of insert (non-template strand) are shown in the top left corner. All insert are 32-nt long and start 252-nt downstream from the promoter. Position of the insert on gel (shown thick black lane) is estimated according to denatured DNA ladders with steps of 10 nucleotides and 100 nucleotides, designated by vertical 10 and 100, respectively. The white block arrow shows the repeat-exiting blockage signal, and the white oval shows diffuse blockage signal; for the supercoiled DNA (right panel), the ‘total’ blockage area is shown by white rectangle. The run-off in the case of linear DNA (left panel) corresponds to the transcript of define length, whereas in the case of supercoiled DNA (right panel), the apparent run-off is likely to be an unresolved mixture of long products of spontaneous transcription termination at multiple sites within the plasmid. The relative intensities of the blockage signals for various sequences are shown on graphs below the respective panels. In these graphs, the intensities of the respective blockage signals were first normalized to the run-off intensities, and then the ratios of these normalized intensities to the normalized intensity of the G32 insert were plotted versus the G-content of the non-template strand of the insert. Each data point is the average of two experimental results, and error bars show deviations from the average. Letters A, T and C designate the non-G base.

Figure 3.

Effect of a nick in the non-template strand on transcription blockage for various sequences under various conditions. Linearized plasmids pN-aga-0, pN-aga-G16, pN-aga-C16 (see Materials and Methods) were used in these experiments. G16 and C16 inserts (from 252 to 267 nt from the promoter) and nick position (after 248 nt from the promoter) are shown by the thick black line and black arrow, respectively. The vertical dotted line at the left shows the approximate area where blockage signals occur.Headings (A, U, G = 1, C = 0.1) and (A, U, G, C = 1) designate ribonucleotide composition during transcription (see Supplementary Materials and Methods). The incorporation of radioactive CTP per transcript is 10-fold larger at A, U, G = 1, C = 0.1 conditions than at A, U, G, C = 1 conditions, which makes radioactive signals in the first case stronger, despite the fact that lowering CTP concentration decreases intensity of transcriptions. Proper normalization and control experiments with radioactive adenosine triphosphate showed that, in fact, at A, U, G = 1, C = 0.1 conditions, the intensity of transcription is ∼3-fold lower than at A, U, G, C = 1 conditions (Supplementary Figure S1).

Figure 4.

Transcription in a mixture of different DNA templates at low and high RNAP/DNA ratios. As a test plasmid, pN-aga-G16 (further referred as G16) was used, which contains the G16 insert with the site for the nicking enzyme localized in the non-template strand 3-nt upstream from the insert. This plasmid produces strong blockage when it contains nick, but <1% blockage without the nick; as a control plasmid, hTel-C was used, which does not produce detectable blockage. The sequence between the promoter and insert (0.25 kb) was identical for control plasmid and G16 plasmid. Both test and control plasmids were linearized by HindIII. To eliminate errors due to product losses during their purification and gel loading, a ‘spiking transcript’ was made in separate transcription reaction from the template pN-aga-hTel-C linearized by DraIII (which also does not produce any detectable blockages) and added to all reactions after transcription was stopped but before purification of transcription products (see Supplemental Materials and Methods for details). Transcription reactions were performed 12 μl at A, U, G, C = 1 conditions (see Supplemental Materials and Methods). For low RNAP/DNA conditions (lanes 1–5), 0.24 units of T7 RNAP and 200 ng of each DNA template were used per one reaction, whereas in high RNAP/DNA (lanes 7–11) conditions, 20 units of T7 and 10 ng of each DNA template were used; thus, T7 RNAP/DNA ratio varied 1670 times between these conditions. The transcription reaction contains either G16-plasmid alone (lanes 1, 2, 7, 8) or control plasmid (lanes 5 and 11), or their equimolar mixture (lanes 3, 4, 9, 10). Lanes 1, 3, 7 and 9 as well as 2, 4, 8 and 10 correspond to G16-plasmid with or without nick upstream of the G16 insert, respectively. To provide convenient intensity for quantitation, the transcription reaction for spiking transcript was also performed at both low RNAP/DNA and high RNAP/DNA conditions, except that the amounts of template were 120 ng and 6 ng, respectively. The spiking reaction was stopped by addition of 3 μl of 100 mM ethylenediaminetetraacetic acid, and then 2 μl of respective spiking transcripts were added to all transcription mixtures after the stop buffer. Also, 2 μl of spiking transcription reaction alone at high RNAP/DNA condition was processed as the rest of the samples and loaded on gel (lane 6). For convenience of visual analysis, a higher exposure (for lanes 1–5) and a lower exposure (for lanes 6–11) of the areas of the gel within dashed-bordered boxes are shown. For the nicked G16 plasmid, well-pronounced blockage signals could be seen. At high RNAP/DNA ratio, the intensities of blockage signals relative to run-off increase; additionally, the distribution of intensities of blockages noticeably shifts in downstream (i.e. towards run-off) direction. For example, the ratio of intensity of the strongest blockage signal (larger white diamond) to the intensity of run-off was about three times larger, and its ratio to the intensity of one of the weaker downstream blockage signals (smaller white diamond) was about two times smaller at high RNAP/DNA conditions than under low RNAP/DNA conditions. The charts show ratios of radioactive signal intensities for various transcripts, further referred to as ‘ratios of transcripts’. All intensities are normalized to the number of cytosines within the transcript; thus, they represent the molar ratios of transcripts. They were also normalized to the intensities of the spiking transcript signals in the same lanes. The data correspond to the average of two experiments, and deviations from the average are shown by the error bars. Results for low RNAP/DNA conditions are shown in black, and for high RNAP/DNA conditions in gray. (A) Radio-autograph of the gel; (B) Ratio of transcripts obtained from G16 plasmid (either with or without nick) in mixture with the control plasmid to the transcripts obtained from the control plasmid in the same mixture. For G16 plasmid without nick, this ratio is close to 1 at both low and high RNA/DNA conditions, which would be expected for two plasmids with similar initiation and elongation rates. For the G16-plasmid with nick, at high RNAP/DNA conditions this ratio is ∼0.2, and at lower RNAP/DNA conditions, it increases up to ∼0.6, but does not reach 1, suggesting that the blockage is at least partially irreversible (See Supplementary Discussion); (C) Ratios of transcripts obtained from the control plasmid in mixture with G16-plasmid to the transcripts obtained from the reaction containing the control plasmid alone. The G16-plasmid used in this experiment is either with a nick (nick +) or without a nick (nick −). At high RNAP/DNA conditions, these ratios are close to 1, consistent with an excess of free RNAP in solution and, consequently, no interference between different templates. At low RNAP/DNA, for the mixture with non-nicked G16 plasmid, this ratio approaches 0.5, which suggests that most RNAP in solution are in the DNA-bound state and are evenly distributed between templates. In the case of nicked G16 plasmid, this ratio is ∼1.6 times smaller, which might indicate some additional RNAP sequestration by the nicked G16 plasmid; (D) Ratio of transcripts obtained from G16-plasmid with nick to transcripts obtained from G16 plasmid without nick. Reactions were performed with each of the templates, separately. This ratio changes ∼3-fold (from 0.13 to 0.38), i.e. the difference between these two templates decreases, on switching from high to low RNAP/DNA conditions.

Figure 5.

Comparison of blockages produced by GGGX repeats. The left panel shows the entire gel, whereas top and bottom right panels show the run-off area with reduced exposure times, and the blockage area with higher exposure, respectively. For linear templates (lanes 1–3), the repeat-exiting blockage signal (white block arrow) is clearly pronounced over the background only for X = A. For supercoiled templates (lanes 4–6), the repeat-exiting blockage signal is pronounced also for X = T, but its value (normalized on ‘run-off’) is 13 times smaller, than for X = A. For X = C, the repeat-exiting blockage signal is not pronounced; however, several blockage signals are evident within the insert. The explanation for this pattern is presently unclear; however, it could be compared with the blockage signals produced by other sequences by estimating the total blockage signal over the area shown by the white rectangle, which includes all blockages. To perform the comparison, a signal was calculated over the same area also for GGGA and GGGT repeats. For GGGT and GGGC repeats, this blockage was seven and three times less than that for GGGA repeats, respectively.

Figure 6.

Possible mechanism for R-loop interference with transcription. (A) The basic mechanism. Normally, the nascent RNA (shown by the thick black line) interacts with certain area (shown by stripped patch) of RNA polymerase (shown by gray oval with dotted line border). R-loop formation (either via thread-back mechanism or some other mechanism shown by dashed line with question mark) disrupts (possibly, partially) this interaction, thus destabilizing the elongation complex and making it more prone to stalling or/and dissociation. (B) Factors that exacerbate the blockage by facilitating R-loop formation: (1) Sequence that forms extra-stable RNA/DNA hybrid (shown by thick patterned line; (2) negative supercoiling, which increases fluctuative opening of DNA; (3) nick in the non-template strand, which decreases propensity of the non-template strand to displace RNA; involvement of the part of the non-template DNA strand (shown by the thick patterned line) in triplex formation with the DNA duplex upstream (4) or downstream (5) of the transcription complex, which would sequester non-template DNA strand thus decrease its propensity to displace RNA. In addition to facilitating R-loop formation, some of these factors could additionally exacerbate blockage by other mechanisms. For example, extra-stable RNA/DNA hybrid inside the transcription complex could interfere with the nascent RNA separation, and triplexes could create obstacles for RNAP movement.