Distinguishing the roles of Topoisomerases I and II in relief of transcription-induced torsional stress in yeast rRNA genes

Abstract

To better understand the role of topoisomerase activity in relieving transcription-induced supercoiling, yeast genes encoding rRNA were visualized in cells deficient for either or both of the two major topoisomerases. In the absence of both topoisomerase I (Top1) and topoisomerase II (Top2) activity, processivity was severely impaired and polymerases were unable to transcribe through the 6.7-kb gene. Loss of Top1 resulted in increased negative superhelical density (two to six times the normal value) in a significant subset of rRNA genes, as manifested by regions of DNA template melting. The observed DNA bubbles were not R-loops and did not block polymerase movement, since genes with DNA template melting showed no evidence of slowed elongation. Inactivation of Top2, however, resulted in characteristic signs of slowed elongation in rRNA genes, suggesting that Top2 alleviates transcription-induced positive supercoiling. Together, the data indicate that torsion in front of and behind transcribing polymerase I has different consequences and different resolution. Positive torsion in front of the polymerase induces supercoiling (writhe) and is largely resolved by Top2. Negative torsion behind the polymerase induces DNA strand separation and is largely resolved by Top1.

FIG. 1.

rRNA genes in top1Δ cells are robustly transcribed and display unusual bubble structures in the active rDNA template. (A) The top panel shows an EM image of ∼20 active rRNA genes from a single yeast nucleolus from top1Δ cells (bar, 0.5 μm). Numbered arrows point to bubble structures in genes. Bottom panel shows each bubble at a higher magnification with an accompanying trace of the DNA strand in which the bubble occurs. (B) Representative rRNA genes from the WT and top1Δ (SY84) strains, with each gene displaying ∼average polymerase density for the strain. Bars (for genes in panels B and C), 0.5 μm. (C) Single rRNA genes from top1Δ cells. The genes in the two panels on top have fewer polymerases than average, with topo-bubbles in some but not all of the polymerase-free gaps (middle gene). The lower gene has high pol density with a small bubble in a small polymerase-free gap. Bubbles 8 to 10 are enlarged at bottom.

FIG. 2.

Topo-bubbles can be distinguished from replication bubbles. (A) Schematic of the tandem rDNA repeat showing position of the origin of replication (r-ARS) and replication fork barrier (RFB) in the nontranscribed spacer (NTS). Shown below this, and aligned in terms of gene-spacer-gene position, are rDNA regions from top1Δ cells displaying either a replication bubble in the NTS (bracketed arrows) or a topo-bubble within and near the 5′ end of the 35S rRNA gene (arrow). Bubbles are enlarged in inset. Bar, 0.5 μm. (B) Examples of replication and topo-bubbles from nearby regions on an EM grid. (C) Examples of longer bubbles of both types. Vertical arrows indicate bubble ends. Replication bubble (top) is ∼10 kb long. Topo-bubbles (bottom) are ∼1.7 and ∼1.35 kb. (D) Plot of bubble length distribution for 147 topo-bubbles and 20 replication bubbles, from SY84 and W1854-2A top1Δ strains.

FIG. 3.

Effect of RNase H under- and overexpression on topo-bubbles in Top1-deficient cells. (A) The left plot shows the topo-bubble frequency in Top1-deficient cells in conditions of RNase H under- and overexpression. For RNase H underexpression, strain YAEH275 (P_GAL-TOP1 rnh1Δ rnh201Δ) was switched from galactose to glucose medium for 6 h to deplete Top1. Its control strain was YAEH271 (P_GAL-TOP1), which was also depleted of Top1 for 6 h. For RNase H overexpression, strain YAEH269 (top1Δ+pGAL-RNH201) was grown in galactose medium for expression of RNH201. Its control strain YAEH267 (top1Δ+pGAL) was similarly grown. Topo-bubble frequency in experimental strains (i.e., Top1-deficient and plus or minus RNase H) is shown as a fraction of the frequency in Top1-deficient control strains; (see the text for the frequency of bubble-containing genes). The bar graph on the right shows the average topo-bubble length (error bars indicate the standard deviation) in YAEH267 compared to YAEH269 using same gene populations used for topo-bubble frequency. (B) Representative rRNA genes from RNase H underexpression experiment in panel A, showing examples with (+) and without (−) topo-bubbles from both strains. Topo-bubbles within genes are enlarged in insets (arrows). (C) Same as panel B for RNase H overexpression experiment.

FIG. 4.

Topo-bubbles appear as regions of DNA helix melting and preferentially occur in AT-rich regions of the gene. (A) Shown at left are portions of four active rRNA genes from top1Δ with topo-bubbles showing typical characteristics: both sides of the bubble appear similar and with no evidence of RNA involvement, even when immediately juxtaposed to RNA polymerases. At the right are portions of two active rRNA genes from top1Δ obtained using modified Miller spreading conditions (KCl concentration increased by 11 mM) (57). In these conditions, DNA in topo-bubbles appears thicker than flanking double-stranded DNA (see the text). Bar, 0.1 μm. (B) Schematic of rDNA repeat at top is aligned with a graph showing the percent AT content across the repeat (middle) and a plot of bubble position across the repeat (bottom). The latter was derived by normalizing gene length to 100 U, dividing each gene into 50 bins of 2 U each, and scoring each bin across the gene as positive or negative (for denatured DNA at that position) for each bubble-containing gene analyzed. n = 101 genes. (C) Gene schematic is aligned with EM of an active rRNA gene with two topo-bubbles (bracketed black arrows). Double-headed red arrows indicate nearest-neighbor RNA polymerases up- and downstream of both bubbles. This gene and 35 additional genes are schematized below the EM image, with each gene normalized in length and with topo-bubble occurrence shown. Nearest-neighbor polymerases up- and downstream of each bubble are shown as red ovals. Shaded bars indicate the three most AT-rich regions, as shown in panel B.

FIG. 5.

Polymerases do not pile up upstream or run off downstream of topo-bubbles. (A) Schematic of analysis in which lengths were measured between bubbles (n = 85) and the nearest polymerases. Average lengths between nearest polymerases and nearest bubble fork are indicated in μm and approximate base pairs. Topo-bubble length (n = 220) from Table 1. (B and C) Relationship between bubble length and distance to nearest upstream (B) or downstream (C) polymerase. (D) Relationship between distance to nearest upstream polymerase and distance to nearest downstream polymerase. (E) Relationship between bubble length and polymerase-free gap size.

FIG. 6.

Evidence that some negative topological stress occurs as melted DNA in top1Δ cells in vivo. (A) Schematic of the two inter-convertible forms of DNA under negative topological stress: melted versus negatively supercoiled. Shown is a hypothetical DNA plasmid of 325 bp, which has a relaxed linking number (Lk₀) of 31 (i.e., 325/10.5 = 31). The linking number (Lk) has contributions from twist and writhe, i.e., Lk = twist + writhe. Relaxed plasmid on left has linking number (Lk) of 31 due to 31 helical twists, and thus its ΔLk = 0, where ΔLk = Lk − Lk₀ (difference between the actual and the relaxed linking number). On the right is a plasmid of the same length but under negative topological stress (ΔLk = −3), which can be relieved by melting a portion of the DNA or by negative writhe, both of which allow maintenance of stable B-form helix in most of the DNA. (B) ChIP analysis of occupancy of single-stranded DNA-binding protein RPA large subunit (encoded by RFA1) over the rDNA repeat and on ACT1 gene. Shown are the relative ChIP signals in control strain YB169 (TAP-RFA1) versus top1Δ strain YB168 (top1Δ, TAP-RFA1) after normalization to the rDNA5 primer within the rRNA gene. Error bars show standard error of the mean for three independent experiments. (C) EM images of several rRNA genes using modified Miller spreading conditions in which formaldehyde is added to cells prior to cell disruption, mimicking conditions used for ChIP fixation. Arrows indicate topo-bubbles in two nearby genes. Bubbles shown enlarged in the inset. Bar, 0.5 μm.

FIG. 7.

Top2 inactivation results in slow elongation in rRNA genes. (A to D) Representative rRNA genes from top2-ts cells at permissive (23°C) and restrictive (35°C for 2 h) temperatures, as labeled. The inset in panel A shows a polymerase density plot comparing polymerases per gene in top2-ts (strain HT2C1A2) at 23°C versus 35°C for 2 h. On average, there were 45 polymerases/gene at 23°C (n = 105 genes) and 57 polymerases/gene at 35°C (n = 113). The difference between these mean values is significant, with a t test P value of 2.6 × 10⁻⁹. (E to H) Representative rRNA genes from top1Δ top2-ts cells at permissive (23°C) and restrictive temperatures (35°C for 2 h), as labeled. Panels A, B, E and F are at the same magnification and show overviews of nucleolar regions, including multiple rRNA genes for each condition (bar, 0.5 μm). Dotted-line brackets in panels A, B, and E (all the same length, equal to that of an active rRNA gene) are aligned with single rRNA genes, each of which is transcribed from 5′ to 3′ end. The same length dotted line is shown in panel F but is not aligned with a gene because in these conditions (Top1 and Top2 depletion) no genes are seen that are transcribed for this length. Instead, individual active genes appear as relatively short regions with very dense polymerase backbones (example, arrowhead in panel F), suggesting that polymerases are unable to traverse the entire gene. Panels C, D, G, and H (at same magnification) show single active rRNA genes for each condition (bar, 0.5 μm). Brackets in panels C and D indicate regions of the gene in which most of the transcripts have been cleaved in an early rRNA processing step (see the text). (The gene in panel D is the same as the bracketed gene in panel B. For improved clarity, Photoshop was used to remove a nearby gene seen in panel B from the image in panel D.) An arrow in panel G indicates a topo-bubble. The gene in panel H is not fully transcribed, as shown by the length of its transcribed region in comparison to fully transcribed genes in panels C, D, and G. This is attributed to poor processivity (see the text).

FIG. 8.

Model for the effects of Top1 and Top2 depletion on Pol I transcription. (A) In WT cells, Top1 and Top2 maintain the rDNA template in a relatively relaxed topological form, as shown by the relaxed DNA between polymerases. WT rRNA genes exhibit a characteristic rRNA processing pattern, consisting of compaction of pre-small subunit RNA into a large 5′-terminal knob (SSU processome), followed by cotranscriptional cleavage to separate small subunit pre-rRNA (released and no longer visible in chromatin spreads) from large subunit pre-RNA (still extruding from the polymerase) (37, 56). (B) In cells deficient in Top2 activity, polymerases are tightly packed on most rRNA genes, and cotranscriptional cleavage is often “advanced” (double-gradient pattern) due to continued processing on dawdling polymerases. Both of these features are characteristic of slow elongation, which is consistent with increased positive torsion (“+” signs) and/or supercoiling in the absence of Top2. Although DNA writhing is not seen in EM spreads, its in vivo presence is inferred (as shown by the positive supercoil) since writhed DNA is the substrate for Top2 (61). (C) In cells deficient for both Top1 and Top2 activity, polymerases are rarely seen to transcribe beyond the first half of the gene, which is consistent with DNA so tightly wound (“+” signs and positive writhe) that it resists the unwinding necessary for transcription (23, 66). (D) In Top1-deficient cells, many genes show no evidence of topological abnormalities, presumably due to the action of Top2 and to local dissipation of transcription-induced positive and negative stress between elongating polymerases (“+” and “−” signs between polymerases). Excess negative torsion (due to more widely spaced polymerases) results in DNA breathing which favors the formation of R-loops (the top gene in panel D). Such R-loops are transient due to endogenous RNase H activity (see reference and results in the present study). In many genes (e.g., the bottom gene in panel D), sufficient negative torsion accumulates to melt the template DNA, particularly in AT-rich regions. Evidence suggests that the melted DNA is bound by RPA (peanut shapes on bubble). (E) When RNase H activity is compromised in Top1-deficient cells (P_GAL-TOP1 rnh1Δ rnh201Δ), topo-bubble formation greatly decreases (Fig. 3), and transcript density increases (23). We propose that undigested R-loops significantly slow Pol I elongation, disallowing the accumulation of hypernegative topological stress and the formation of topo-bubbles.