From the journal RSC Chemical Biology Peer review history

12-Nov-2020

Dear Professor Min:

Manuscript ID: CB-ART-10-2020-000192
TITLE: Light-induced modulation of DNA recognition by the Rad4/XPC damage sensor protein

Thank you for your submission to RSC Chemical Biology, published by the Royal Society of Chemistry. I sent your manuscript to reviewers and I have now received their reports which are copied below.

I have carefully evaluated your manuscript and the reviewers’ reports, and the reports indicate that major revisions are necessary.

Please submit a revised manuscript which addresses all of the reviewers’ comments, including the additional experiments requested. Further peer review of your revised manuscript may be needed. When you submit your revised manuscript please include a point by point response to the reviewers’ comments and highlight the changes you have made. Full details of the files you need to submit are listed at the end of this email.

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I look forward to receiving your revised manuscript.

Yours sincerely,
Dr Andrea Rentmeister

************

Reviewer 1

This is a fairly interesting paper on incorporation of N3-photocaged T into DNA duplex and study of interaction with Rad4/XPC - it is probably not the top 10% but still interesting and publishable in RSC ChemBiol. The N3-photocaging of T creates a mismatch which recruits the Rad4/XPC protein - not surprising but worthwhile to confirm. After photochemical uncaging the protein does not bind. The problem is that the photochemical experiments were only done with the DNA itself (in absence of protein) - the experiments must be remade with irradiation of the mixture of caged-DNA and protein - to rule out the possibility of photocross-links! The fluorescence study of the conformation changes is interesting. I would recommend acceptance of this work after major revision and adding two key experiments which may improve the work:
1. the authors only studied the EMSA of photocaged and uncaged DNA with different concentrations of the protein - however, even more important experiment would be to do EMSA with the mixture photocaged DNA and the protein with different times of the irradiation (of the mixture) - which should decrease the interaction with the protein (unless there are some photochemical cross-links - which need to be rules out).
2. there were some recent works using visible light for release of NB or nitropiperonyl-photocaging in DNA or RNA - the authors should try 400 or 405 nm light which is less toxic and less prone to formation of photocross-links. See:
https://doi.org/10.1039/C9SC00205G
https://doi.org/10.1002/anie.202013936

Reviewer 2

I enjoyed reading this manuscript by Min et al. very much. They picked a very interesting subject in combining light-activation strategies with the DNA repair story - which has not been done very often, yet. I can imagine quite a number of applications - some of which are suggested in the manuscript - and would know what to do with these findings right away, myself. The paper is rich in methodology - in some traditional and some new ways -  which itself is helpful for the community. However, there are some very few aspects that I very strongly propose to address: please do not use the terms "photoswitching" and "irreversible" together. "Photoswitching" is the reversible approach and "uncaging"/"phototriggering" the irreversible one. This is how everybody else in the community does it. Also, photochemical conversion does not proceed via an exponential function and hence there is no such thing as a photochemical half-life time. This term is applicable to monomolecular thermal conversions or radioactive decay. The correct formula is the one of a "stretched exponential". People often do not notice when fitting because their data is often noisy - covering the discrepancy. But deriving numeric data using the wrong analytical function is simply wrong.


Both aspects can be easily corrected in no time. After that I believe that the manuscript is ready for publishing in RSC. Chem. Biol.

December 14, 2020

Dear Dr. Rentmeister

Thank you for your email and encouraging responses to our manuscript. We have revised our manuscript to accommodate the suggested changes and included point-by-point responses to each reviewer’s comment below in blue text. The Discussion is highlighted in its entirety because we had rearranged the paragraphs to improve clarity.

We look forward to hearing your favorable decision. Thank you very much again.

Best regards,
Jung-Hyun Min

========================

12-Nov-2020

Dear Professor Min:

Manuscript ID: CB-ART-10-2020-000192
TITLE: Light-induced modulation of DNA recognition by the Rad4/XPC damage sensor protein

Thank you for your submission to RSC Chemical Biology, published by the Royal Society of Chemistry. I sent your manuscript to reviewers and I have now received their reports which are copied below.

I have carefully evaluated your manuscript and the reviewers’ reports, and the reports indicate that major revisions are necessary.

Please submit a revised manuscript which addresses all of the reviewers’ comments, including the additional experiments requested. Further peer review of your revised manuscript may be needed. When you submit your revised manuscript please include a point by point response to the reviewers’ comments and highlight the changes you have made. Full details of the files you need to submit are listed at the end of this email.

Please submit your revised manuscript as soon as possible using this link:

*** PLEASE NOTE: This is a two-step process. After clicking on the link, you will be directed to a webpage to confirm. ***

https://mc.manuscriptcentral.com/rsccb?URL_MASK=5be66ecc194c4300acbc154afe1d0c6d

(This link goes straight to your account, without the need to log on to the system. For your account security you should not share this link with others.)

Alternatively, you can login to your account (https://mc.manuscriptcentral.com/rsccb) where you will need your case-sensitive USER ID and password.

You should submit your revised manuscript as soon as possible; please note you will receive a series of automatic reminders. If your revisions will take a significant length of time, please contact me. If I do not hear from you, I may withdraw your manuscript from consideration, and you will have to resubmit. Any resubmission will receive a new submission date.

Supporting our community through Covid-19
While our publishing services are running as usual, we also know that this is a very challenging time for everyone, for many different reasons. If any aspect of the publishing process is worrying you – for example you think you may struggle to meet a pre-determined deadline – please let us know, and we will work out an answer together.

The Royal Society of Chemistry requires all submitting authors to provide their ORCID iD when they submit a revised manuscript. This is quick and easy to do as part of the revised manuscript submission process. We will publish this information with the article, and you may choose to have your ORCID record updated automatically with details of the publication.

Please also encourage your co-authors to sign up for their own ORCID account and associate it with their account on our manuscript submission system. For further information see: https://www.rsc.org/journals-books-databases/journal-authors-reviewers/processes-policies/#attribution-id

Please note: to support increased transparency, RSC Chemical Biology offers authors the option of transparent peer review. If authors choose this option, the reviewers’ comments, authors’ response and editor’s decision letter for all versions of the manuscript are published alongside the article. Reviewers remain anonymous unless they choose to sign their report. We will ask you to confirm whether you would like to take up this option at the revision stages.

I look forward to receiving your revised manuscript.

Yours sincerely,
Dr Andrea Rentmeister

************
We are grateful to the reviewers for their time and thoughtful comments. Our responses are in blue in this letter and the changes in the text are marked with yellow highlight.

REVIEWER REPORT(S):
Referee: 1

Comments to the Author
This is a fairly interesting paper on incorporation of N3-photocaged T into DNA duplex and study of interaction with Rad4/XPC - it is probably not the top 10% but still interesting and publishable in RSC ChemBiol. The N3-photocaging of T creates a mismatch which recruits the Rad4/XPC protein - not surprising but worthwhile to confirm. After photochemical uncaging the protein does not bind.
The problem is that the photochemical experiments were only done with the DNA itself (in absence of protein) - the experiments must be remade with irradiation of the mixture of caged-DNA and protein - to rule out the possibility of photocross-links! The fluorescence study of the conformation changes is interesting. I would recommend acceptance of this work after major revision and adding two key experiments which may improve the work:
1. the authors only studied the EMSA of photocaged and uncaged DNA with different concentrations of the protein - however, even more important experiment would be to do EMSA with the mixture photocaged DNA and the protein with different times of the irradiation (of the mixture) - which should decrease the interaction with the protein (unless there are some photochemical cross-links - which need to be rules out).
We thank the reviewer for the comment. Indeed, it would be important to confirm the absence of any unwanted nonspecific photodamage or photochemical crosslink in the system as a side effect of uncaging irradiation. In Figures 5A and S10, we had in fact shown the fluorescence conformational studies of the DNA-protein complexes after irradiating them together. The results showed that NPOM-DNA in the presence of Rad4 reverts to the B-DNA form similar to the unmodified DNA after > 2 min of irradiation. This observation thus also indicated that the DNA is no longer specifically bound to protein after the co-irradiation.

These experimental results corroborate with the notion that decaged DNA is nonspecific DNA and protein doesn’t bind specifically to it. However, it does not completely rule out if nonspecific photocrosslinks still formed in a manner that was not detectible by the FRET probes as incorporated in the current FLT experiments. To address this, we have carried out additional EMSA experiment, as suggested. The protein-DNA complexes were prepared as before, but before running on EMSA, we irradiated each protein-DNA complex samples for 2 mins on ice. The results show that the level of the NPOM-DNA bound to Rad4 indeed decreased with increased irradiation, down to a level shown in the nonspecific control sample. Light irradiation did not alter the level of the unmodified DNA nonspecifically bound to Rad4. In sum, these results show that the light irradiation leading to NPOM-DNA photocleavage in our experiments does not induce nonspecific protein-DNA photocrosslinks. These results are now included as a new Figure S4.
2. there were some recent works using visible light for release of NB or nitropiperonyl-photocaging in DNA or RNA - the authors should try 400 or 405 nm light which is less toxic and less prone to formation of photocross-links. See:
https://doi.org/10.1039/C9SC00205G
https://doi.org/10.1002/anie.202013936
We thank the reviewer for the insightful suggestion. As per the suggestion, we have irradiated NPOM-dT and NPOM-DNA using 405 nm light and indeed we observed irradiation time-dependent change in the absorption spectra, consistent with the photocleavage of NPOM adduct. The kinetics of photocleavage appeared similar as with the previous setting using 365 nm. The result is now included as a new Figure S2.

Referee: 2

Comments to the Author
I enjoyed reading this manuscript by Min et al. very much. They picked a very interesting subject in combining light-activation strategies with the DNA repair story - which has not been done very often, yet. I can imagine quite a number of applications - some of which are suggested in the manuscript - and would know what to do with these findings right away, myself. The paper is rich in methodology - in some traditional and some new ways - which itself is helpful for the community. However, there are some very few aspects that I very strongly propose to address: please do not use the terms "photoswitching" and "irreversible" together. "Photoswitching" is the reversible approach and "uncaging"/"phototriggering" the irreversible one. This is how everybody else in the community does it.
We thank the reviewer for the comments and advice. We have revised the terms ‘photoswitch’, as suggested. The changes are marked with yellow highlights in the manuscript.
Also, photochemical conversion does not proceed via an exponential function and hence there is no such thing as a photochemical half-life time. This term is applicable to monomolecular thermal conversions or radioactive decay. The correct formula is the one of a "stretched exponential". People often do not notice when fitting because their data is often noisy - covering the discrepancy. But deriving numeric data using the wrong analytical function is simply wrong. Both aspects can be easily corrected in no time. After that I believe that the manuscript is ready for publishing in RSC. Chem. Biol.
We thank the reviewer for the comment and suggestion. As stretched exponential fitting needed clear saturation points in the data, we first re-measured the absorption spectra for the NPOM-DNA and NPOM-dT photocleavage reactions over a longer time course than before (increased from 240 sec to 720/1200 seconds). Then, we fit the data using Abs(t)=〖A+B*ⅇ〗^(-(t/t_0 )^b ) where b was fixed as 1 for the simple single exponential decay but was used as a fit parameter for the stretched exponential decay1. The results for 365 nm irradiation for NPOM-DNA and NPOM-dT are shown below:



Overall, we consistently see that the stretched exponential in fact generate fits that have b>1, which indicates “squeezed exponential” rather than stretched exponential1. Moreover, the fits using the stretched exponential function are only marginally better than the single-exponential function despite the fact that the stretched exponential has an additional fit parameter. When we constrained the b parameter to fall between 0 and 1 (i.e., stretched exponential), the fits consistently gave b = 1.
Furthermore, in our reading of the literature, we were unable to find a stretched exponential function describing the time dependence of the number of molecules that are photoconverted as a function of time, for the case when the photoconversion is irreversible, as it is in our study. Adapting the description of the photoconversion reaction of azobenzene 2 and the photodissociation of ligand from the heme group in hemoglobin 3 to our system, we can write a simple irreversible kinetic scheme N→N^* for the photoconversion that we are observing, where N^* is the species after photoexcitation. In this case, the rate of change of N^* concentration may be written as (dN^*)/dt=kN where the apparent rate coefficient k is proportional to the product of the intensity of the excitation light, the probability that it gets absorbed (which depends on the extinction coefficient at the excitation wavelength), and the probability that the molecule will get converted if it absorbs a photon (the quantum yield). The solution to the above equation is a single-exponential decay from N to N^*. While the actual photoconversion process (the photochemistry) itself may be a stretched exponential, that process occurs on much faster time scales than the seconds conversion that we are observing.
Therefore, we have retained the use of single exponential decay fitting for our data in the manuscript. Please also note that the detailed kinetics and mechanisms of the photoreaction are outside the scope of this paper although they are very interesting problems to address. We hope that we have provided a satisfactory explanation for the choice of the exponential function and that our current analyses are acceptable to the reviewer.
References:
1. J. Sworakowski and K. Matczyszyn, Acta Physica Polonica A, 2007, 112, S153
2. I. Mita, K. Horie and K. Hirao, Macromolecules, 1989, 22, 558-563.
3. W. A. Saffran and Q. H. Gibson, Journal of Biological Chemistry, 1977, 252, 7955-7958.

18-Dec-2020

Dear Professor Min:

Manuscript ID: CB-ART-10-2020-000192.R1
TITLE: Light-induced modulation of DNA recognition by the Rad4/XPC damage sensor protein

Thank you for submitting your revised manuscript to RSC Chemical Biology. After considering the changes you have made, I am pleased to accept your manuscript for publication in its current form. I have copied any final comments from the reviewer(s) below.

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With best wishes,

Dr Andrea Rentmeister

Reviewer 1

the revised version is now suitable for acceptance