Whether the DNA relaxation activities of eukaryotic topo I and topo II are redundant or have distinctive roles during genome transactions is a long-standing question. In this study, we show that the sole inactivation of topo II in S. cerevisiae
produces a decrease of virtually all Pol II transcripts of length above ~3
kb, irrespective of their function. This selective reduction is not due to an alteration of transcription initiation, but to an impairment of the RNA polymerase during elongation. Therefore, proper production of this subset of transcripts demands a specific role of topo II, which cannot be compensated for by topo I.
Any dependence of transcript abundance on transcript length can be reasoned by the alleged role of topoisomerases in removing the DNA torsional stress generated during transcription elongation (3
). In vitro
studies have shown that the failure to relax (+) torsional stress stalls the progression of transcribing polymerases (6
). The same has been concluded from recent in vivo
work, which showed that convergent transcription is impaired under topo I and topo II inactivation as a function of supercoiling accumulation (8
). Hence, a dependence on transcript length is expected in cells deficient in topoisomerase activity, since the longer the transcribed distance the more (+) torsional stress will be accumulated. Strikingly, we do not observe such length dependence in Δtop1 top2-ts
double mutants of S. cerevisiae.
Inactivation of both topoisomerases causes instead a general reduction of transcript abundance, which probably reflects a global down-regulation of transcription by alterations in both the initiation and elongation steps, irrespective of the gene size (12
). Our observation is consistent with a recently reported intragenic distribution of Pol II in Δtop1 top2-ts
double mutants of S. cerevisiae
, which showed no significant length dependence alterations relative to wild-type strains (15
Similar results have been found for Δtop1 top2-ts
double mutants of S. pombe
; in which only a slight accumulation of Pol II was observed in coding regions of genes (12
Contrasting with the above, the transcript length dependence uncovered here is prominent and occurs specifically in top2-ts
single mutants. No such dependence is observed in Δtop1
single mutants. The inactivation of topo II does not affect the transcription frequency of long genes. It produces a stall in the advancement of Pol II after transcribing beyond 2
kb, which suitably explains the selective decrease of long transcript abundance. The transcript reduction is not rescued by overproducing yeast topo I or bacterial topo I, which relaxes (−) DNA supercoils. It is rescued by catalytically active topo II or a GyrBA enzyme, which relaxes (+) DNA supercoils. Therefore, the more likely cause of the stall of Pol II and subsequent reduction of long transcripts is the incapacity of the top2
cells to relax the (+) DNA supercoils accumulated during transcription elongation. This finding substantiates the biological relevance of what we had observed in previous in vitro
studies: (+) supercoiled chromatin is efficiently relaxed by the DNA cross-inversion mechanism of topo II, but not by the DNA strand-rotation mechanism of topo I (16
An intriguing aspect of the transcript length dependence exposed here is that, rather than being gradual, it has a sharp inflection point at ~3
kb. Up to this critical length, (+) torsional stress can be either relaxed by topo I alone or it is not high enough to delay the normal progression of Pol II. Beyond this length, topo I is no longer efficient and topo II is then required to avoid the stalling of Pol II. We believe that this inflection reveals how increasing levels of (+) torsional stress affect the conformation of intracellular chromatin. In this regard, in vitro
studies had shown that torque generated ahead of RNA polymerases is enough to partially unwrap nucleosomal DNA and flip from (−) to (+) the entry and exit DNA crossing of the nucleosomes (18–20
). This plasticity allows eukaryotic chromatin to buffer moderate levels of (+) torsional stress without shortening or buckling supercoils. However, when this buffering capacity is surpassed, chromatin enters into a supercoiling regime (18–20
). At this stage, chromatin tightly condenses and (+) torsional stress may enforce also a flip of the global left-handed chirality of nucleosomes (18
). Remarkably, we had previously showed that chromatin is efficiently relaxed by topo II but not by topo I when its DNA superhelical density is >0.04 (16
), a value in which chromatin has entered the supercoiling regime (18–20
). With these premises, the abrupt reduction of long transcript production reported here is likely to reflect the transition of intracellular chromatin from the buffering stage to the supercoiling stage during transcription elongation, as illustrated by the model in . This transition probably occurs during normal transcription, since the generation rate of torsional stress in front of RNA polymerases is higher than the relaxation rate provided by cellular topoisomerases (19
). Accordingly, we propose that topo I activity alone and the buffering capacity of yeast chromatin are sufficient to support the normal production of transcripts up to ~3
kb in length. After this point, increasing levels of (+) torsional stress drive chromatin into the supercoiling regime downstream of the transcription complex. Now, topo I is no longer efficient and the relaxation of DNA by topo II activity becomes essential to support the normal progression of the RNA polymerase.
Figure 6. Model to explain the specific role of topo II during transcription elongation in long genes. At transcription initiation, the typical nucleosome organization of downstream chromatin stabilizes (−) DNA supercoils. During transcriptional elongation, (more ...)
Our conclusions on the requirement of topo II activity during Pol II elongation are comparable to those inferred for Pol I transcription of yeast rDNA genes. Former studies indicated that inactivation of either topo I or topo II stimulates Pol I transcription initiation; whereas inactivation of both enzymes affected the elongation of long, but not short, Pol I transcripts (14
). More recent visualization analyses revealed that effective elongation by Pol I depends mainly on topo II activity (17
). Therefore, although Pol I and Pol II markedly differ in the structure and dynamics of their transcribing ensembles, topo II may be similarly required for the removal of (+) DNA supercoils accumulated ahead of both RNA polymerases. The biological relevance of this activity is probably masked by the other essential roles of topo II during chromosome replication and segregation (4
). Since topo II inactivation decreases the abundance of long transcripts but does not abolish them, incipient or minor phenotypes are possibly undetected along the other major or lethal effects of topo II.
In prokaryotes, DNA topoisomerases have a clear division of roles to relax DNA during transcriptional elongation. Bacterial topo I specifically relieves (−) torsional stress, whereas DNA gyrase removes (+) supercoils (3
). In eukaryotes, however, since topo I and topo II are able to relax both (+) and (−) torsional stress, a general belief is that both enzymes can substitute for each other in these tasks. Our study demonstrates that this is not the case. Topo II has a specific role in removing (+) supercoils accumulated during transcriptional elongation, a function that cannot be compensated by cellular topo I. These findings imply also that biological and pharmacological effects of topoisomerase inhibitors could be dependent on local levels of DNA supercoiling and, thus, on the transcript length of neighbouring genes.