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Topological stress is responsible for the detrimental outcomes of head-on replication-transcription conflicts

02 Aug 2019-bioRxiv (Cold Spring Harbor Laboratory)-pp 691188
TL;DR: It is found that head-on conflict resolution requires the relaxation of positive supercoils by type II topoisomerases, DNA gyrase and Topo IV, and it is suggested that gyrases plays a fundamental role in the evolution of head- on genes.
Abstract: Conflicts between replication and transcription machineries have profound effects on chromosome duplication, genome organization, as well as evolution across species. Head-on conflicts (lagging strand genes) are significantly more detrimental than co-directional conflicts (leading strand genes). The source of this fundamental difference is unknown. Here, we report that topological stress underlies this difference. We find that head-on conflict resolution requires the relaxation of positive supercoils by type II topoisomerases, DNA gyrase and Topo IV. Interestingly, we find that after positive supercoil resolution gyrase also introduces excessive negative supercoils at head-on conflict regions, driving pervasive R-loop formation. The formation of these R-Loops through gyrase activity is most likely caused by the diffusion of excessive negative supercoils through RNA polymerase spinning. Altogether, these results provide critical mechanistic insights into head-on replication-transcription conflicts and suggest that gyrase plays a fundamental role in the evolution of head-on genes.

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.

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Topological stress is responsible for the detrimental outcomes of head-on
replication-transcription conflicts
Kevin S. Lang
1
and Houra Merrikh
1#
1
Department of Biochemistry
Light Hall
Vanderbilt University
Nashville, TN
#
Corresponding author and lead contact
Houra Merrikh
Email: houra.merrikh@vanderbilt.edu
Tel: 615-343-3846
.CC-BY-NC-ND 4.0 International licensea
certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under
The copyright holder for this preprint (which was notthis version posted August 2, 2019. ; https://doi.org/10.1101/691188doi: bioRxiv preprint

Lang and Merrikh
Abstract
Conflicts between the replication and transcription machineries have profound effects on
chromosome duplication, genome organization, as well as evolution across species. Head-on
conflicts (lagging strand genes) are significantly more detrimental than co-directional conflicts
(leading strand genes). The source of this fundamental difference is unknown. Here, we report
that topological stress underlies this difference. We find that head-on conflict resolution requires
the relaxation of positive supercoils DNA gyrase and Topo IV. Interestingly, we find that after
positive supercoil resolution, gyrase introduces excessive negative supercoils at head-on
conflict regions, driving pervasive R-loop formation. The formation of these R-Loops through
gyrase activity is most likely caused by the diffusion of negative supercoils through RNA
polymerase spinning. Altogether, our results address a longstanding question regarding
replication-transcription conflicts by revealing the fundamental mechanistic difference between
the two types of encounters.
1
.CC-BY-NC-ND 4.0 International licensea
certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under
The copyright holder for this preprint (which was notthis version posted August 2, 2019. ; https://doi.org/10.1101/691188doi: bioRxiv preprint

Lang and Merrikh
Introduction
Transcription and DNA replication occur simultaneously on the same template. The lack of
spatiotemporal separation between these two processes leads to conflicts between them every
replication cycle. Replication and transcription machineries can encounter each other either
head-on or co-directionally. Co-directional conflicts occur when genes are transcribed on the
leading strand whereas head-on conflicts occur when genes are transcribed on the lagging
strand. It has been demonstrated that head-on conflicts are more deleterious than co-directional
conflicts in that they cause increased mutagenesis, DNA breaks, replisome stalling and restart,
(Lang et al. 2017; Paul et al. 2013; Million-Weaver et al. 2015; Million-Weaver, Samadpour, and
Merrikh 2015; Mirkin and Mirkin 2005; Prado and Aguilera 2005; French 1992; J. D. Wang,
Berkmen, and Grossman 2007; C. N. Merrikh and Merrikh 2018; H. Merrikh et al. 2011;
Pomerantz and O’Donnell 2010; Hamperl et al. 2017). Despite many insightful studies into these
inevitable encounters, the fundamental question regarding why head-on conflicts are more
detrimental than co-directional conflicts remains unanswered. It is perplexing that encounters
between the same two machineries (the replication machinery or the replisome, and RNA
polymerase or RNAP) can have such different outcomes simply due to orientation.
Topological constraints could explain why head-on conflicts are more deleterious than
co-directional conflicts. Unwinding of DNA during transcription generates positively supercoiled
DNA ahead, and negatively supercoiled DNA behind RNAP (Wu et al. 1988; Liu and Wang
1987). Similarly, during replication, positive supercoils accumulate in front of the replisome (Vos
et al. 2011; Postow, Peter, and Cozzarelli 1999; Hiasa and Marians 1996). The resolution of this
supercoiled DNA is critical for both transcription and replication to proceed efficiently (Khodursky
et al. 2000). In a co-directional conflict, the positive supercoiling generated in front of the
replisome would encounter the negative supercoiling produced from active RNAPs ahead. This
would most likely cause a net neutral change in local supercoiling levels. However, during a
head-on conflict, the positive supercoiling generated ahead of the replisome would encounter
the positive supercoiling produced by RNAP. Therefore, in a head-on conflict, there may be a
transient buildup of positive supercoils that has the potential to change the fundamental
mechanics of the replisome and RNAP. Such changes could stall the replisome, leading to
disassembly, and changing the dynamics of RNAP and associated mRNAs. These predictions
suggest that torsional stress could be the key driver of conflict severity and therefore this model
must be tested.
Another key question is whether topoisomerases are critical conflict resolution factors. The
resolution of supercoils in all organisms requires topoisomerases (Champoux 2001; J. C. Wang
2002; Vos et al. 2011). In bacteria, there are two topoisomerases that relax positive supercoils:
DNA gyrase and Topo IV. DNA gyrase and Topo IV are both required for replication fork
progression in vivo (Khodursky et al. 2000; Crisona et al. 2000; Peng and Marians 1993; Ashley
et al. 2017; Vos et al. 2011). Topo IV also plays a critical role in the resolution of catenanes
(intertwined chromosomes) as well as the separation of sister chromatids during segregation
(Hiasa and Marians 1996; Zechiedrich and Cozzarelli 1995). If the torsional stress hypothesis is
2
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certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under
The copyright holder for this preprint (which was notthis version posted August 2, 2019. ; https://doi.org/10.1101/691188doi: bioRxiv preprint

Lang and Merrikh
correct, then type II topoisomerases should be critical conflict resolution factors, yet, this
question has not been addressed.
Here, we report that type II topoisomerases preferentially associate with head-on genes and
that cells harboring engineered head-on conflicts are sensitized to type II topoisomerase
inhibitors. Accordingly, we find that conditional depletion of either gyrase or Topo IV is
deleterious to cells experiencing engineered head-on conflicts. 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. Remarkably, however, we find
that negative supercoil introduction by DNA gyrase at head-on conflict regions is responsible for
the formation of toxic R-loops at these regions. Consistent with this finding, we 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. Furthermore, an allele of gyrase that is strongly defective in introduction of
negative supercoils completely rescues the lethality of cells lacking RNase HIII that are
experiencing head-on conflicts. This rescue is observed in experiments examining both
engineered and endogenous head-on conflicts, which arise predominantly during the expression
of stress response genes.
Results
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. In B.
subtilis
, relaxation of positive supercoils is accomplished by the activity of either gyrase or Topo
IV (Vos et al. 2011; Postow, Crisona, et al. 2001; Crisona et al. 2000; Ashley et al. 2017). If the
model of positive supercoil accumulation at head-on conflict regions is correct, then these
enzymes should preferentially associate with a head-on conflict region. To test this hypothesis,
we measured gyrase and Topo IV enrichment genome-wide, using chromatin
immunoprecipitation followed by deep sequencing (ChIP-Seq). Most head-on genes are not
expressed under standard laboratory conditions. Rather, the majority of head-on genes are
induced under specific conditions, such as during exposure to environmental stress (Nicolas et
al. 2012; Mostertz et al. 2004; Guariglia-Oropeza and Helmann 2011; Lang et al. 2017).
Therefore, we did not expect to see enrichment of type II topoisomerases at endogenous
head-on genes during growth in rich media. In order to study the effects of topology at head-on
conflict regions, we took advantage of several different tightly controlled engineered conflict
systems, all of which were integrated onto the chromosome. In each of these systems the same
exact gene (e.g. lacZ
) was inserted onto the chromosome in the same locus, in either the
head-on or co-directional orientation with respect to replication. To control for gene expression
levels, both the head-on and co-directional version of each gene was placed under the control
of the same promoter (e.g. P
spank(hy)
).
3
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certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under
The copyright holder for this preprint (which was notthis version posted August 2, 2019. ; https://doi.org/10.1101/691188doi: bioRxiv preprint

Lang and Merrikh
In order to measure the relative association of type II topoisomerases with the conflict regions,
we used a GFP fusion to the GyrA subunit of gyrase (Tadesse and Graumann 2006) and
constructed a 3xMyc fusion to the ParC subunit of Topo IV. We expressed an IPTG-inducible
lacZ
gene in either the head-on or the co-directional orientation, and performed ChIP-Seq
experiments in order to obtain a high resolution map of the association of type II
topoisomerases with the engineered conflict regions. We found that both gyrase and Topo IV
are preferentially enriched at the engineered conflict locus when the orientation of lacZ
is
head-on (Figure 1). Importantly, this enrichment was transcription-dependent. When we
measured enrichment of these topoisomerases using ChIP-qPCR, we found that in the absence
of the inducer, IPTG, the levels of topoisomerases at the engineered conflict regions were
similar in the two orientations (Supplementary Figure 1). Furthermore, we 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. It is noteworthy that we utilized standard
formaldehyde crosslinking for the GyrA ChIPs. However, we were unable to ChIP ParC using
formaldehyde. The ParC association was only detectable when we performed the ChIP assays
using ciprofloxacin crosslinking, which specifically crosslinks active type II topoisomerases on
DNA.
Inhibition of type II topoisomerases increases replisome enrichment/replication stalling
at head-on but not co-directional genes
In E. coli
, gyrase and Topo IV promote replication fork progression (Khodursky et al. 2000). 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. We
tested this hypothesis by performing ChIP-seq of the replisome protein, DnaC, as a proxy for
replication stalling. If fork progression is unimpeded, the distribution of DnaC enrichment should
be equal along the genome in asynchronous bacterial cultures. We have demonstrated
previously that DnaC enrichment is a good proxy for replication fork stalling (Lang et al. 2017; H.
Merrikh et al. 2011; C. N. Merrikh, Brewer, and Merrikh 2015). To inhibit type II topoisomerase
activity, we used subinhibitory doses of the antibiotic novobiocin. Novobiocin is a competitive
inhibitor of type II topoisomerase ATPase activity (Sugino et al. 1978; Hardy and Cozzarelli
2003; Maxwell 1993). We performed ChIP-Seq experiments, where we measured the
association of DnaC with the engineered conflict regions in media with and without sublethal
concentrations of novobiocin (375 ng/mL). In untreated cells, we found preferential association
of DnaC with the engineered conflict region in the head-on orientation (Figure 2, top panel).
When the cells were treated with novobiocin, there was an increase in DnaC enrichment at the
head-on but not the co-directional conflict region. These results suggest that without type II
topoisomerase activity, topological problems at head-on genes can impede replication.
4
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certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under
The copyright holder for this preprint (which was notthis version posted August 2, 2019. ; https://doi.org/10.1101/691188doi: bioRxiv preprint

Citations
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Abstract: Rapid improvements in sequencing and array-based platforms are resulting in a flood of diverse genome-wide data, including data from exome and whole-genome sequencing, epigenetic surveys, expression profiling of coding and noncoding RNAs, single nucleotide polymorphism (SNP) and copy number profiling, and functional assays. Analysis of these large, diverse data sets holds the promise of a more comprehensive understanding of the genome and its relation to human disease. Experienced and knowledgeable human review is an essential component of this process, complementing computational approaches. This calls for efficient and intuitive visualization tools able to scale to very large data sets and to flexibly integrate multiple data types, including clinical data. However, the sheer volume and scope of data pose a significant challenge to the development of such tools.

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Frequently Asked Questions (11)
Q1. What is the effect of novobiocin on 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. 

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. 

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. 

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. 

Given that gyrase activity is facilitating R-loop formation, their results suggest that the activity of this enzyme leads to increased mutagenesis, albeit indirectly. 

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. 

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. 

Most importantly, their previous work demonstrated that the increased mutagenesis of head-on genes is driven by R-Loops in wild-type cells. 

The “transcription off” control for this engineered conflict is achieved through the use of a strain where this promoter is constitutively off. 

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. 

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).