Peer review of: Persistent telomere cohesion protects aged cells from premature senescence (2020)




Reviewers' comments: 

Reviewer #1 (Remarks to the Author): 

Thank you to the authors for effectively addressing all my queries. I support publication of the revised 
manuscript. 

Reviewer #2 (Remarks to the Author): 

The authors have made efforts to address some of the concerns raised previously, however several 
main issues remain outstanding. This dampens one’s enthusiasm to endorse it for publication as it 
currently stands. 

1. The TRF1AA work cited by the authors demonstrates that the tankyrase binding site of TRF1 is 
responsible for the observed phenotypes (resolution of persistent telomere cohesion, subtelomere 
recombination) in aged cells overexpressing TRF1WT, but it does not demonstrate the causal 
relationship between the phenotypes. As a result, the authors have not convincingly shown that there 
is a causal relationship among phenotypes caused by overexpression of TRF1WT in aged cells. In the 
absence of experimental evidence for a demonstrated causal relationship between persistent telomere 
cohesion and subtelomere recombination, their conclusion that persistent telomere cohesion protects 
against inappropriate recombination is not sufficiently supported. 

2. The authors showed that overexpression of TRF1WT in U2OS cells induces DNA damage and cellular 
senescence, which is in stark contrast to the published report by Silva et al that showed that 
overexpression of TRF1WT in U2OS does not affect cell cycle profile. The authors attributed this 
discrepancy to the difference in their ways of doing experiments, e.g. through Day 5 vs Day 7 by Silva 
et al. It is rather inadequate that the authors just stated that “we cannot say what the FACS analysis 
would look like at Day 7”. Given that their data in Fig. 5O showed that 30% of TRF1WT-overexpressed 
cells are senesced, it is difficult to imagine that the cell cycle profile of these cells on Day 7 would be 
similar to control cells. The authors should at least make the effort to perform FACS analysis (a 
method that they have used in the manuscript) on Day 5 and Day 7 to clarify this discrepancy. 
The discrepancy between their results and the published results begs a concern as to whether an 
excessive amount of overexpressed TRF1WT in U2OS cells (rather than resolution of persistent 
telomere cohesion per se) is responsible for causing subtelomere recombination, DNA damage and 
rapid growth arrest. This concern was not addressed by the authors but needs to be experimentally 
addressed (e.g. knocking down endogenous TRF1 but not overexpressed TRF1WT in U2OS cells), 
especially given that the authors have not demonstrated a direct link that resolution of persistent 
telomere cohesion causes subtelomere recombination. 

3. The authors provided their measurement on the frequency of APBs in their rebuttal. They should 
also cite these APB data and discuss the associated concern in the manuscript. 

4. The authors declined to address whether overexpression of TRF1WT causes increased DNA damage, 
RAD51 foci and growth arrest in BIBR-treated HCT75 PD245 cells. A major concern in this manuscript 
is about the effect of overexpressed TRF1WT on various phenotypes including DNA damage and 
growth arrest (see the point #2 above). Therefore, it is critical that the authors address this issue to 
substantiate their findings. 

Reviewer #3 (Remarks to the Author): 

The authors have appropriately addressed my comments in their entirety.