Topological stress is responsible for the detrimental outcomes of head-on replication-transcription conflicts
Summary (3 min read)
Introduction
- Transcription and DNA replication occur simultaneously on the same template.
- During a head-on conflict, the positive supercoiling generated ahead of the replisome would encounter the positive supercoiling produced by RNAP.
- Inhibition of type II topoisomerase activity leads to increased stalling of the replisome when it approaches a gene transcribed in the head-on, but not the co-directional orientation.
Type II topoisomerases preferentially associate with a head-on but not a co-directional engineered conflict region
- The relaxation of both positive and negative supercoils is an essential process in all cells.
- Most head-on genes are not expressed under standard laboratory conditions.
- When the authors measured enrichment of these topoisomerases using ChIP-qPCR, they found that in the absence of the inducer, IPTG, the levels of topoisomerases at the engineered conflict regions were similar in the two orientations .
- Furthermore, the authors confirmed that the GyrA signal was specific by performing control ChIPs of GFP only (unfused to GyrA) and found no enrichment at the lacZ gene in either orientation.
- The authors were unable to ChIP ParC using formaldehyde.
Inhibition of type II topoisomerases increases replisome enrichment/replication stalling at head-on but not co-directional genes
- If torsional stress is a major problem at head-on conflict regions, then subtle inhibition of these topoisomerases should lead to increased replication fork stalling at head-on conflict regions.
- The authors have demonstrated previously that DnaC enrichment is a good proxy for replication fork stalling (Lang et al.
- To inhibit type II topoisomerase activity, the authors used subinhibitory doses of the antibiotic novobiocin.
- The authors performed ChIP-Seq experiments, where they measured the association of DnaC with the engineered conflict regions in media with and without sublethal concentrations of novobiocin (375 ng/mL).
Sublethal amounts of novobiocin compromises cell survival specifically in the presence of a strong head-on conflict
- The authors previously showed that in the absence of critical conflict resolution factors, head-on conflicts can significantly compromise survival efficiency (Lang et al.
- To test this hypothesis, the authors measured survival efficiency using colony forming units (CFUs) of cells containing the engineered conflicts, in the head-on or the co-directional orientation, upon chronic treatment with various concentrations of novobiocin.
- When the cells were plated on novobiocin, again, there was no difference in survival efficiency between cells carrying the head-on or co-directional lacZ when transcription was off.
- The authors performed the survival experiments with this system as described above.
Both gyrase and Topo IV are critical for the resolution of head-on conflicts
- Novobiocin has activity against both gyrase and Topo IV, although the affinity of the drug for Topo IV is much weaker than that for gyrase (Peng and Marians 1993; Sugino et al. 1978) .
- It can't be ruled out that Topo IV activity is also inhibited to some extent under these conditions.
- To directly determine the contribution of each of the two enzymes to conflict resolution, the authors adapted a conditional degradation system (Griffith and Grossman 2008) to specifically deplete the GyrB subunit of gyrase or the ParC subunit of Topo IV.
- In order to detect potentially subtle differences in survival of their engineered conflict strains, the authors used concentrations of IPTG that only slightly depleted GyrB, and subtly impacted survival of wild-type cells (gyrase is essential, so a complete depletion cannot be used here).
- The authors then tested the survival of cells carrying engineered conflicts under these conditions, but now the engineered conflicts expressed lacZ from a different promoter, P xis , which is constitutively active.
Inhibition of type II topoisomerases reduces R-Loop formation at head-on conflict regions
- There is evidence in the literature that topoisomerase activity can influence R-Loop formation, at least in vitro and in human cells (Massé and Drolet 1999; Tuduri et al. 2009 ) .
- The authors performed DNA-RNA Hybrid ImmunoPrecipitations coupled to deep sequencing (DRIP-Seq) experiments using the S9.6 antibody, which recognizes RNA:DNA hybrids.
- Consistent with what the authors have measured previously using qPCR (Lang et al. 2017) , they found more R-loops when the lacZ gene was expressed in the head-on orientation compared to the co-directional orientation .
- Remarkably, the authors found that when type II topoisomerases are inhibited, R-loop levels are reduced at head-on conflict regions.
Inhibiting type II topoisomerases rescues R-Loop mediated replisome stalling
- If type II topoisomerase activity is driving R-loop formation at head-on genes, then treating cells with low doses of novobiocin should reduce replisome stalling at head-on conflict regions in cells lacking RNase HIII.
- The authors tested this hypothesis using DnaC ChIP-Seq, as described above.
- As the authors published previously, they found that there is a preferential association of DnaC with head-on versus co-directional conflict regions, and this difference is significantly increased in cells lacking RNase HIII .
- When the authors treated cells with low amounts of novobiocin to inhibit topoisomerase activity, there was a marked decrease in DnaC enrichment at the head-on conflict region .
Inhibiting type II topoisomerases rescues death by R-Loops
- If topoisomerase activity is driving R-loop formation at head-on genes, then limiting that activity should increase the viability of cells that contain an engineered head-on conflict and lack RNase HIII.
- The authors tested this model by measuring the viability of cells lacking RNase HIII, and expressing either the head-on or co-directional lacZ in the presence of low concentrations of novobiocin.
- As expected, cells with the co-directional reporter had no growth defect when the lacZ gene was induced with IPTG.
- Remarkably, chronic novobiocin exposure rescued these defects in a dose dependent manner .
- Altogether, these results suggest that the resolution of head-on conflicts by type II topoisomerase activity is driving toxic R-loop formation.
Introduction of negative supercoils by gyrase promotes toxic R-Loop formation at head-on conflict regions
- Novobiocin inhibits both gyrase and Topo IV activity.
- Therefore, even if the positive supercoil relaxation activity of gyrase is impacted by the R138L mutation, the major effect of this mutation at the conflict region will be a loss of negative supercoil introduction.
- As expected, there was no effect of transcription on the viability of the cells carrying the co-directional reporter construct.
- Remarkably, the authors found that the gyrB R138L mutation completely rescued this lethality .
- These results demonstrate that it is specifically the introduction of negative supercoils by gyrase at head-on conflict regions that leads to the formation (and/or stability) of toxic R-loops.
Discussion
- The authors results strongly suggest that positive supercoils build up at head-on conflict regions.
- Alternatively, the sudden release of torsional strain by type II topoisomerases could cause RNAP to rapidly progress, generating excessive negative supercoils and R-loop formation (Kuzminov 2017) .
- Here, the authors find that R-loop formation and/or stabilization at head-on genes stems from the introduction of negative supercoils by gyrase at these regions.
- The authors previously proposed that the head-on orientation is retained for some genes as a mechanism to increase mutagenesis and promote gene specific evolution (Paul et al.
- The authors discovered (what appears to be) the main source of gene orientation-specific problems in replication-transcription conflicts.
Did you find this useful? Give us your feedback
Citations
2,187 citations
77 citations
33 citations
32 citations
14 citations
References
10,798 citations
2,513 citations
"Topological stress is responsible f..." refers background in this paper
...The resolution of supercoils in all organisms requires topoisomerases (Champoux 2001; J. C. Wang 2002; Vos et al. 2011)....
[...]
...DNA supercoiling is regulated by topoisomerases (Champoux 2001; J. C. Wang 2002; Vos et al. 2011)....
[...]
2,194 citations
"Topological stress is responsible f..." refers background in this paper
...DNA supercoiling is regulated by topoisomerases (Champoux 2001; J. C. Wang 2002; Vos et al. 2011)....
[...]
2,084 citations
1,766 citations
"Topological stress is responsible f..." refers background in this paper
...However, this unwinding causes subsequent changes in DNA supercoiling both upstream and downstream of both machineries (Liu and Wang 1987; Wu et al. 1988)....
[...]
Related Papers (5)
Frequently Asked Questions (11)
Q2. What are the contributions in "Topological stress is responsible for the detrimental outcomes of head-on replication-transcription conflicts" ?
Here, the authors report that topological stress underlies this difference. Interestingly, the authors find that after positive supercoil resolution, gyrase introduces excessive negative supercoils at head-on conflict regions, driving pervasive R-loop formation. 1. CC-BY-NC-ND 4. 0 International license a certified by peer review ) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity.
Q3. What is the effect of limiting topoisomerase activity?
If topoisomerase activity is driving R-loop formation at head-on genes, then limiting that activity should increase the viability of cells that contain an engineered head-on conflict and lack RNase HIII.
Q4. How many L of protein A beads were added to the GE?
After incubation with the antibody, 40 µL of 50% Protein A sepharose beads (GE) were added and IPs were incubated at 4° C for 2 hours with gentle rotation.
Q5. What is the role of gyrase in causing mutagenesis?
Given that gyrase activity is facilitating R-loop formation, their results suggest that the activity of this enzyme leads to increased mutagenesis, albeit indirectly.
Q6. What is the effect of inhibition of type II topoisomerases on cell survival?
If type II topoisomerases are indeed important for conflict resolution, then the inhibition of these enzymes should impact survival of cells experiencing head-on conflicts.
Q7. What is the main reason why head-on genes gain beneficial mutations faster than if they?
Under selection, these head-on genes will likely gain beneficial mutations faster than if they were co-directionally oriented, simply due to the increased mutation rates which are facilitated by conflicts.
Q8. What is the main reason for the increased mutagenesis of head-on genes?
Most importantly, their previous work demonstrated that the increased mutagenesis of head-on genes is driven by R-Loops in wild-type cells.
Q9. How is the transcription off control achieved?
The “transcription off” control for this engineered conflict is achieved through the use of a strain where this promoter is constitutively off.
Q10. What is the effect of the type II topoisomerases on head-on conflict?
Consistent with this finding, the authors observe that, in cells lacking the RNase HIII enzyme, which resolves R-Loops, inhibition of type II topoisomerases lowers R-loop abundance, and alleviates R-Loop induced replisome stalling at head-on genes.
Q11. Why is gyrase able to reduce the number of negative supercoils?
This is likely due to the introduction of negative supercoiling by gyrase, asLang and Merrikhnegatively supercoiled DNA will energetically favor R-loop formation, although recent work has suggested that highly positively increased supercoiling could also impact R-Loop formation (Stolz et al. 2019).