Peer review of: Topoisomerase 1 prevents replication stress at R-loop-enriched transcription termination (2020)

Reviewers' Comments: 
 
Reviewer #1: 
Remarks to the Author: 
 
In this manuscript, Promonet, Padioleau, Liu et al use ChIP-seq to map the distribution of R-loops, 
phospho-RPA (pRPA) and γ-H2AX around transcribed genes in HeLa cells, both under normal 
conditions and during mild depletion of Top1. Interestingly, the authors find that although R-loops 
are enriched at both the transcription start site (TSS) and transcription termination site (TTS), 
pRPA is specifically enriched at TTS. Additionally, the enrichment of R-loops and pRPA is 
substantially elevated at convergent genes that are actively transcribed. When Top1 is depleted, γ-
H2AX is enriched around sites bound by pRPA, suggesting that Top1 acts to prevent the formation 
of double-strand breaks at these sites. The authors extend this observation by performing BLESS 
to analyze the distribution of DSBs: DSBs are enriched at TSS, but are dramatically increased at 
TTS upon Top1 depletion in about a quarter of genes. The authors synthesize these data into a 
model whereby replication forks encountering RNA polymerase head-on at TTS stall and activate 
ATR, thereby preventing further replication into the gene. When Top1 is depleted, the resulting 
topological stress leads to fork collapse and DSBs 
 
Overall I think this is a nicely executed study that advances the field enough to warrant publication 
in Nature Communications with few revisions. The authors make careful and interesting 
observations, which come together into a coherent model that is not only consistent with recent 
literature, but also extends the conceptual framework of how TRCs and R-loops impact DNA 
replication. I do not think additional experimental work is required before publication, and my only 
real criticism is that the manuscript feels a bit too condensed. It’s hard to intuit exactly how some 
of the analyses were done, even after thorough and repeated readings (e.g. what is the basis of 
the five quintiles in figure 3b? How was the clustering done for Figure 4?). The authors should 
ensure that the overall workflow is a bit easier to follow. 
 
 
 
Reviewer #2: 
Remarks to the Author: 
In this manuscript, Pasero and colleagues address the question of whether all R-loops are created 
equal in their ability to cause DNA damage, using Top1 knockdown as a tool to enhance the 
formation of R-loops at certain genomic sites. Consistent with previous reports, their data suggest 
that R-loops form predominantly at transcription start sites (TSS) and transcription termination 
sites (TTS), and that Top1 knockdown enhances R-loop formation specifically at TTS. Using pS33 
RPA, a marker of ATR activation, and gammaH2AX, a marker of ATR, ATM and DNAPK activation, 
they then go on to show that the head-on convergence of a replication fork with a TTS leads to 
replication stress, marked by pRPA. In the absence of Top1, problems resulting from such 
convergence are enhanced and forks seem to collapse, leading to DSB formation. This is supported 
by the increased phosphorylation of H2AX and increases in DSB formation at TTS sites where fork 
convergence occurs. 
 
This is an exciting manuscript that sheds new insight into a very important question in R-loop field 
about the similarities and differences in R-loops. It also helps address the question of how R-loops 
mechanistically cause damage. Using a variety of genomic approaches, the authors demonstrate 
that just a subset of R-loops are causing problems for the cell -- those where forks converge head 
on with transcription termination sites. They also go on to restate the model as one in which 
transient fork pausing occurs at sites of transcription and R-loop formation, to ultimately help 
suppress a collision. Generally I think this is a very well done study and the data are interpreted 
carefully. It is quite appropriate for Nat Comm and should be of broad interest. However, there are 
some relatively minor issues that should be addressed before publication, and I have a few 
suggestions that will strengthen the work. 

 
Specific Comments: 
The authors should in some way validate the formation of DSBs in their system. Both H2AXp and 
RPAp (on this site) can be replication stress markers and neither is specific to DSBs. It would be 
helpful to show by comet assay or another approach (e.g. with a more DSB specific marker) that 
breaks are forming under the shTOP1 conditions used here. 
 
A related/overlapping point is whether the authors can show that the increased H2AX resulting 
from Top1 knockdown is really due to break formation and ATM activation at TTSs. Does an ATM 
inhibitor and not ATR inhibitor reduce this signal; or is a more break-specific marker induced at 
some of these sites? 
 
A few sites of RPAp and hybrid formation at the TTS of several converging genes with and without 
Top1 should be validated using qPCR approaches. A related point is that the authors should 
validate that DRIP signal is up using qPCR at the TTS (and not over the whole gene) upon Top1 
knockdown. 
 
The authors should include a scatter plot (or some other genome-wide analysis) to compare R-
loops after Top1 knockdown and before. Do all sites go up or are some down as well? 
 
Related to the last point, my understanding is that Manzo et al (2019) didn’t observe the same 
differential effects at the TSS and TES upon TOP1 depletion if I understand that work correctly. 
Given that one of the authors here is an author on both papers they must be aware of this and 
should address the potential differences. 
 
There is little explanation or speculation of why Top1 might be particularly important in preventing 
breaks at the TES. I think it would strengthen the manuscript to come back to this in the 
discussion in a little more detail. 
Please compare RPA sites with and without Top1 knockdown – it’s not clear if the old sites are the 
same as the new one or if the sites are new after knockdown. 
 
Figure 2a and d – is this all with Top1 knockdown? How does it compare to signal in control cells. 
Figure 2d should include this comparison and a screen shot (for 2a) comparing results with and 
without Top1 would be helpful. 
 
It’s unclear how the clustering in Figure 4 was done - there appear to be a number of positive 
regions in their “negative” cluster 2, and a number of negative regions in their “positive” cluster 1 
in the control samples, so it is assumed that they only took into account the siTOP1 condition 
when doing this clustering. It seems surprising to perform clustering when you only end up with 2 
categories (vs multiple dimensions). Why not just rank them from most positive to least positive, 
and set some sort of threshold? If these clusters represent distinct categories of genes (i.e. 
induced in TOP1 vs decreased), the names of the clusters should reflect this or at least be more 
informative. 
 
The significance of some of the H2AX data may be overstated or overinterpretated. To this 
reviewer it appears high in general over genic regions when Top1 is knocked down. Additionally, in 
the metaplots shown in 3B, the signal doesn’t seem to resolve to the site of the TTS very well. It 
might help to show, for example, that there is no accumulation around the closest TSS to each 
observed TTS. Alternatively, the authors could potentially aggregate signal over a broader window 
(say 200kb on each side)? Related to this, in figure 4, why do the levels of pH2AX seem to be so 
much higher in both categories of genes in the shTOP1 condition. These data suggest that there is 
nothing particularly special about the TTS in terms of pH2AX. Based on these concerns, I think it 
the authors make too much about the H2AX data and may want to soften their language, 
particularly since the BLESS data shows a much higher-resolution and more convincing picture of 
damage at these sites. 

 
Are the places where DSBs are increased by iBLESS the same places where forks converge on TTS 
in a close range? 
 
Could the authors provide error bands on the metaplots (e.g. Figure 1g, 2d, 4b). 
 
Minor points: 
Figure S1b – are these samples all with shTop1? Or control? 
Is the data in Figure S2a the same as that in 2b, top panel