Peer review of: Zuo1 supports G4 structure formation and directs repair toward nucleotide excision repair (2020)

Reviewers' Comments: 
 
Reviewer #1: 
Remarks to the Author: 

In this manuscript De Magis et al. describe, for the first time, a functional protein (Zuo1) that 
promotes G-quadruplex formation to preserve genomic stability in yeast. This is the first example 
that describes a functional and positive biological effect mediated by G-quadruplex DNA secondary 
structures, which is highly novel. Furthermore, the conclusions made by the authors are the result 
of a systematic and rigorous series of biological experiments. Hence, there is no doubt that this 
manuscript should be considered for publication in Nature Communication. 
 
I have, however, a couple of concerns that I feel the authors should address prior publication: 
 
1) From the way the authors interpret their results and also as mentioned a couple of times in the 
manuscript, it seems that Zuo1 is a G-quadruplex "inducer" and that the formation of these 
secondary structures at certain genomic loci stimulates NER activity. Although the authors have 
demonstrated that Zuo1 can bind to G4s biophysically, it would be good to show that this protein 
could actually stimulate/induce G4-formation in a non-folded sequence, to support this hypothesis, 
this can be done for example by CD. Also if Zuo1 is a G4-inducer, why there is no increase of G4-
binding peaks in the Zuo1-overexpressed experiment? The authors should discuss this. 
 
2) The second concern is still related to the first one. Authors can rescue yeast growth in Zuo1 
deficient cells by addition of a G4-ligand such as PhenDC3, which confirms the function of the 
protein to promote G4-formation. However, they still speculate that Zuo1 binding to G4 is key to 
recruit Rad23 but with no evidence to support this directly. Is this really the case? Or is simply 
Zuo1 creating G4s that recruit the NER pahtway on theirs own right? This seems to be more likely, 
otherwise how could PhenDC3 rescue yeast growth in cells that are still deficient of Zuo1? If the 
protein binding to G4 is key to recruit NER PhenDC3 alone could not rescue the phenotype. This 
aspect should be discussed more and caveated in the manuscript prior publication. 
 
 
 
Reviewer #2: 
Remarks to the Author: 
This work describes a potentially interesting discovery linking the protein Zuo1 to G4 structures 
and nucleotide excision repair. By discovering that Zuo1 recognizes G4 structures and allows the 
recruitment of NER proteins, the authors suggest that this mechanism allows the NER mechanism 
to work. The authors' results are convincing and clearly demonstrate that Zuo1 recognizes G4 and 
recruits NER proteins. It remains however that the most interesting part of the authors' model, 
that is to say the repair of UV lesions mediated by the G4 structure formation recognized by Zuo1, 
remains a hypothesis that is not demonstrated directly in this paper. The absence of this 
demonstration makes the paper less attractive, lacking a bit of consistency for a journal like 
Nature Communications. 
I recognize that it may be difficult to demonstrate this hypothesis, but it would be interesting to 
know what fraction of UV damage lesions are repaired by this mechanism in a cell? Could the 
authors demonstrate a delay in eliminating UV lesions in the absence of Zuo1 (using Dot Blot or 
even better XR-seq approach)? 
 
Minor details 
1- To find the protein that directly binds to Zuo1 on G4 in order to recruit NER factor would 
increase the impact of the work. 
 
2- The experiments of ChIP and DOT blot indicate that the absence of Zuo1 leads to a decrease 
amount of the G4 motifs. The authors conclude that Zuo1 supports their formation but can they 
exclude the hypothesis that the presence of Zuo1 simply prevents their resolutions? 
 
3- I would advice to the authors to combine figure 3-b and 3-d to avoid repetition of the wt and 
Zuo1 lanes in these two panels. 
 
4- It would be important to include sgs1d and pif1-m2 yeast strains alone in experiments 3-c-d 
rather than only refering to published papers.