Peer review of: Warsaw Breakage Syndrome associated DDX11 helicase resolves G-quadruplex structures to (2010)

Reviewers' comments: 
 
Reviewer #1 (Remarks to the Author): 
 
DDX11/ChlR1 and its yeast orthologue Chl1 are ATP-dependent DNA helicases related to Xpd and 
FANCJ, that unwinds DNA with 5’-3’ directionality. Mutations in DDX11 are linked with Warsaw 
breakage syndrome (WABS) which is characterised by high incidence of chromosomes with 
centromeric cohesion defects (“railroads”) and “premature chromatid separation” (PCS). It has 
previously been documented that yeast Chl1 and mammalian DDX11 are associated with the 
replisome and promote establishment of sister chromatid cohesion during S phase. How they are 
recruited to the replisome is not fully understood. DDX11 associates with Timeless via a highly 
conserved amino acid motif embedded in a large CHL1/DDX11-specific loop inserted into the 
helicase domain (Cortone et al PLOS gen 2018). Mutations  in DDX11 that abolish this interaction 
show cohesion defects similar to DDX11 depletion. It was recently shown that DDX11 and Timeless 
collaborate in order to detect and process G4 structures during replication (Lerner, LK et al 
bioRxiv). How DDX11 contributes to cohesion establishment and DNA replication is currently 
unclear. 
There is some conflicting information about the requirement of the helicase activity of DDX11/Chl1 
in promoting cohesion establishment. Studies of the yeast and Mammalian Chl1 proteins claim that 
the Walker A mutant (K48 in yeast and K50 in vertebrates) as well as the Q loop mutant Q23, both 
of which abolish helicase activity are fully capable of promoting cohesion establishment (Samora et 
al 2016 and Cortone et al 2018). This has led to the notion that the helicase activity is not required 
and that a hitherto mysterious activity that requires ‘engagement’ with cohesin and/or Scc2 is 
instead the mechanism by which Chl1 promotes cohesion establishment. However, analysis of the 
same Walker mutant in yeast Chl1 or chicken DDX11 by different groups showed the opposite, 
namely that the helicase activity was critical for cohesion (Holloway S 2000 and Abe T et al 2016). 
A recent report also showed that the DDX11 Q23A mutant is incapable of rescuing the cohesion 
defects in WABS fibroblasts, suggesting that the helicase activity is required. Strangely, this paper 
is from the same lab represented in this manuscript (Faramarz A et al PLOS one 2020). This issue 
is an important one, as a requirement for the helicase activity would imply that the helicase 
promotes cohesion establishment by altering the conformation of DNA. The role of Chl1/DDX11’s 
helicase activity therefore is of substantial interest for the cohesin field. 
 
In the current study, the authors present clinical features of seven new WABS patients and 
characterise the new cases initially by identifying bi-allelic mutations in these patients. They show 
cells derived from patients display significant cohesion defects and importantly these defects can 
be corrected by expression of wild type DDX11. A few of the mutations identified are hypomorphic, 
presumably because of their low protein stability (which they measure). Two of the mutations 
(G57R and C705Y) lie in the helicase domain of DDX11, the C705Y mutant is shown to abolish 
DDX11 helicase activity. The paper describes the generation of DDX11 deletion cell lines (with and 
without p53) and shows that this is accompanied by severe cohesion defects. They show that 
overexpression of wild type and the hypomorphic mutants rescue the cohesion defects of the 
DDX11 knockout lines. In contrast, overexpression of the G57R and C705Y mutants as well as the 
K50A Walker A mutant fail to rescue the cohesion defects. These observations are striking and 
support the notion that the helicase activity is necessary for its role in DNA replication/repair and 
in cohesion establishment. Having said this, would it not have been better to ‘knock in’ the mutant 
proteins? If the manuscript had been presented in this manner, it would have warranted 
publication in Nature communications. However, in its current state we cannot recommend 
publication of this manuscript. 
 
The main problem the manuscript suffers from is that too many unnecessary details are presented. 
Most of these (Sensitivity to various drugs, modest reduction in fork speeds, accumulation of 
damage foci) were already know for WABS cells and the observations presented here do not 
greatly alter our understanding. Large sections Figures 1-7 could easily be presented as 
supplements and each discussed with a few lines. The main message of the paper (as prominently 

stated in the title) appears in Figures 7& 8. Any general reader would have to navigate through all 
the details presented with different cells and drugs and redundant messages to get to the heart of 
the story causing them to lose interest. Inexplicably, the evidence that the newly identified 
mutation is defective in helicase activity is presented supplementary Figure S9. We do not see the 
point of cramming all these details into the manuscript just for the sake of adding ‘substance’ to 
the paper, it is very distracting and makes it difficult to read. 
 
Another major issue is the fact that the same group of authors have very recently published a 
paper with all the same assays and show in that manuscript that the helicase activity of DDX11 
(based on the analysis of a Q loop mutant Q23A) is required for cohesion and DNA 
replication/repair (Faramarz A et al PLOS one 2020). Why make two papers out of it? This is a 
matter for consideration by the editor. 
 
The manuscript does have an important message. If this manuscript is to be considered further, it 
needs serious rearrangement and rewriting in emphasizing the main message, namely the 
requirement of the helicase activity for cohesion establishment. The mutant proteins could be 
characterised biochemically to show the effect on ATP binding and hydrolysis in order to make a 
strong case that the helicase activity of DDX11 is important for promoting cohesion establishment. 
 
 
 
Reviewer #2 (Remarks to the Author): 
 
Summary: 
Researchers identified new compound heterozygous mutations linked to Warsaw Breakage 
Syndrome. The DDX11 proteins encoded by the variants display reduced protein stability; the 
corresponding cells are hyper-sensitive to topoisomerase and PARP inhibitors, show defects in 
sister chromatid cohesion, and reduced replication fork speed. DDX11 depletion causes 
chromosomal instability. Treatment of DDX11-deficient cells with G4-interactive compounds was 
reported to impairs growth induce chromosomal instability and cohesion defect to a more 
significant extent than loss of FANCJ, a previously determined FANCJ G4-resolving helicase sharing 
sequence homology with DDX11 in its helicase core domain. DDX11 helicase core domain is 
required for G4 ligand resistance. Based on their experimental results, authors conclude that 
DDX11 is important for protection against G4-induced DSBs as well as cohesion defects that are 
linked to DNA replication. 
 
 
Critical Comments: 
As detailed in the Summary, this is an investigation of the molecular defects of new patients 
afflicted by Warsaw Breakage Syndrome, a DDX11 DNA helicase disorder. The new mutations 
identified help to solidify the molecular pathology underlying the chromosomal instability disorder. 
The evidence supports the conclusions made by the authors, and the experimental studies are 
solid. The results will provide value to researchers and clinicians alike. Statistical analyses are 
appropriate. Experimental procedures and rational are explained well. 
 
A number of concerns are raised below that need to be carefully considered and addressed by the 
authors: 
 
Introduction: Suggest that phrasing “putatively moderate symptoms of human WABS patients” be 
changed as this is subjective and not well defended. 
 
I am a bit confused over the diagnosis of WABS03 as “undoubtedly a WABS patient” (Discussion). 
Based on protein expression it is stated that the corresponding mutant protein R140Q encoded by 
a mutant allele shows a small increase and that evidence it is the disease-causing mutation remain 
limited (paraphrase, page 7 lines 215-219).  Further, immunofluorescence studies suggested that 

the R140Q mutant localized to nucleus similar to wt DDX11. Please clarify the basis for assigning 
WABS03 as a patient with Warsaw Breakage Syndrome so that it is easier for the reader to 
understand. 
 
Perhaps I am missing it, but were all the DDX11 missense mutant proteins, (e.g., R140Q) tested 
for helicase activity in vitro? I see in Figure S9 that C705Y was tested. The authors should test 
R140Q. 
 
On page 6, the authors suggest that the rationale to assess if WABS cells show a replication defect 
as assessed by DNA fiber assays was that DDX11 is known to interact with replication factors. 
While this may be true, a number of these proteins are also implicated in DNA repair pathways. 
Moreover, a previous paper published in 2016 demonstrated that DDX11-deficient cells display 
reduced replication fork progression in cells exposed to the nucleotide-depleting agent 
hydroxyurea compared to DDX11-proficient cells (as demonstrated by DNA fiber studies). I found 
this reference (39) in the Discussion, but perhaps it should be mentioned earlier, as this reference 
(39) provides a stronger rationale for investigating if the lymphoblasts of newly identified DDX11 
patients display replication defects. 
 
Two G4-stabilizing compounds are used for cellular studies: Quarfloxin and CX5461 (reference 53). 
I do not find in reference 53 any mention of Quarfloxin. Perhaps I am missing it. That paper does 
list CX3543 in addition to CX5461. Further reading in the literature revealed to me that Quarfloxin 
is indeed CX3543. Perhaps this could be better spelled out for the reader. 
 
The greater Quarfloxin sensitivity of WABS cells compared to FANCJ-deficient cells is interesting 
but seems under-explored in this study. Although one could argue that the focus of the current 
work is on DDX11, I found the analysis by comparison to be perfunctory. For example, why not 
compare the sensitivity of FANCJ- versus DDX11-deficient cells to telomestatin as well? The 
suggestion that the difference in Quarfloxin sensitivity is based on the observation that “different 
G4 interacting compounds appear to selectively bind different types of G-quadruplexes” 
(demonstrated in vitro) may or may not be relevant in vivo and at first glance seems to be 
speculative. From an experimental standpoint, measuring drug sensitivity by colony survival and 
acute DNA damage induction by an appropriate immunofluorescent marker (e.g., gamma-H2AX; 
53BP1) may provide greater insight than the IncuCyte assay of cell growth for differential effects 
of the G4 stabilizer as it relates to DDX11 versus FANCJ status. 
 
Returning to the point made by the authors that the apparently differential effects of Quarfloxin 
versus Telomestatin on DDX11-deficient cells versus FANCJ-deficient cells may reflect differences 
in G4 binding specificity, the authors should address this directly in the Discussion by reporting 
what has been published in the literature on this issue. The mention in the Discussion that TMS 
stabilizes formation of telomeric G4 structures whereas Quarafloxin selectively targets a G4 
structure commonly found in ribosomal DNA and in promoter sequences, and that quarfloxin 
accumulates in nucleoli. These points raise the question if a particular G4 architecture is favored 
for the ribosomal DNA repeats housed in the nucleoli versus the telomeric repeats of telomeres? 
Given that DDX11 was previously reported to efficiently resolve 5’ ssDNA tailed two-stranded anti-
parallel G4 substrates in vitro but act poorly on unimolecular G4 substrate which FANCJ is capable 
of resolving efficiently, it raises the question following the Reviewer’s comments if Quarafloxin 
preferentially binds bimolecular G4 substrates versus unimolecular G4 substrates, or (as the 
authors also suggest) that the preferential subcellular localization of Quarafloxin to nucleoli plays a 
role and suggests a more prominent nucleolar role of DDX11. Is it known what is the predominant 
G4 architecture of ribosomal DNA repeats? 
 
Given 1) the potential effects of G4-stabilizing compounds on nuclear DNA replication, 2) the 
observation made in the current study that DDX11-deficient cells show reduced replication speed, 
and 3) the previously published result that forks are preferentially slowed by hydroxyurea (an 
agent that like G4 binding cmpds induces replication stress but by a distinct mechanism) in 

DDX11-deficient cells compared to Wild-type cells, I would have expected the authors to perform 
additional DNA fiber analyses to assess effects of the G4-stabilizing agents on not only replication 
fork speed, but other fork dynamic parameters, i.e., fork asymmetry, nascent strand degradation, 
fork restart, etc. These additional studies may have yielded insight to the relationships among 
DDX11 status, G-quadruplexes, chromosomal instability and sister chromatid cohesion, as 
suggested by the authors. Moreover, the DNA fiber studies with DDX11-deficient and proficient 
cells exposed to G4-binding compounds need to be done to support the conclusions made in the 
study and provide greater insight to the apparent G4 ligand sensitivity reported in this manuscript. 
 
In Figure 2 (panels C and D), the authors show a single image of gamma H2AX and DNA fiber 
assays with double label (ongoing replication fork), respectively. The authors should show 
representative images for both panels C and D from the various cell lines in which quantitative 
data are shown. 
 
Similarly, in Figure 3H, representative metaphase spreads for the analyzed cell lines in which 
quantitative data are shown should be depicted. 
 
 
 
 
Reviewer #3 (Remarks to the Author): 
 
Faramarz and colleagues describe molecular and cellular defects in cells derived from patients with 
Warsaw Breakage Syndrome (WBS). Previous studies (as well as the current work) link defects in 
DDX11 to the development of WBS. While mRNA levels are unaffected WBS cells exhibit DDX11 
protein instability. Mutations in some patients are also likely to impair the helicase activity of 
DDX11. In addition to chromosome breakage WBS cells exhibit impaired chromosome cohesion, 
hyper-sensitivity to DNA damaging agents such as camptothecin and PARP inhibitors, decreased 
DNA synthesis and elevated chromosomal aberrations. RPE1 cells knocked out for DDX11 also 
exhibit impaired cell proliferation that is dependent on P53. Importantly they discount the 
contribution of the pseudogene DDX12p to these phenotypes. Faramarz et al go on to show that 
DDX11 defective cells are hyper-sensitive to G4-DNA stabilizing compounds which inhibit cell 
proliferation, induced loss of cohesion and caused increased chromosomal breaks. The same is 
true for WBS lymphoblasts and the defects were reversed in cells expressing wild type DDX11. 
 
This is a nice piece of work, particularly strong when describing the defects associated with loss of 
DDX11 and with WBS. However, it is much less strong on mechanism. It documents defects in 
cohesion and in replication but reports little as to why this is. The biggest limitation is that the 
manuscript highlights the role of DDX11 in protecting against G-quadrupled induced DNA damage 
but little about the relevance of this to normal metabolism. A major limitation is the use of G4-
stabilizing ligands such as pyridostatin. This compound has recently been shown to trapping of 
Topoisomerase 2 on DNA but it is not clear whether this is linked to G4 stabilization 
(https://www.biorxiv.org/content/10.1101/845446v1).  So it is possible that the DDX11 defective 
cells are mainly sensitive to trapping of topoisomerases rather than DNA damage induced by 
persistent G4-DNA structures. Even, so the investigation into the contribution of DDX11 to DNA 
repair and the DNA damage response is quite superficial, particularly as the title emphasizes this 
aspect. It would be useful if the authors looked in more detail at the genetic relationship between 
DDX11 and genes shown to affect response to G4-DNA such as FANCJ, REV1, BLM and WRN. 
Ultimately the reader would like to understand wto which physiological pathway(s) DDX11 
contributes.