The study of protein recruitment to UV-induced DNA lesions can be distorted by photoconversion of DNA dyes like Hoechst or DAPI

A=Authors, R=Reviewer

A: Thank you for sharing your expertise. Your comments helped to significantly increase the quality of the article.
 
R: “UV-induced lesions”
In the title and the text of this report the authors use a term “UV-induced DNA lesions”. The use of this term is misleading, since UV-induced lesions are generally defined as pyrimidine dimers and photoproducts. However, the authors refer to DNA lesions inflicted by 405nm light (which is not UV, see below) in the presence of a DNA-bound fluorescent dye Hoechst, and this leads to induction of a host of various lesions, not only “UV-induced”. When Hoechst 33342 has been introduced into live cells and exposed to 405nm focused laser light, it is (marginally) excited and acts as a photosensitizer. This leads to induction of oxidative damage and DNA breaks, i.e. not typical UV-induced lesions.

A: We added information on the types of damage generated at different wavelengths +- photosensitizing agents.
 
R:“UV laser”
The term ‘UV laser’ is used throughout the manuscript. This term appears incorrect, since the authors used a laser emitting 405nm wavelength light. By definition, 405 nm is visible light (and indeed it is readily visible by human eye, as opposed to UVA, UVB or UVC). I suggest to refer to the Toptica laser, which the authors used, as ‘blue laser”, as in the paper [Kong et al. 2009] which the authors cite.

A: You are right. We corrected that.
 
R: Hoechst photosensitization vs DNA labeling
Fluorescent DNA probe Hoechst is described in two disguises in this paper – as a photosensitizer and as a counterstain. These two roles and the ensuing problems are not clearly distinguished and explained in the paper. If Hoechst or DAPI are added to live cells prior to microirradiation with a focused beam of light of 405nm wavelength, the dye acts as a photosensitizer (as correctly stated in Results). However, in the Abstract the authors state that “the measurements are performed in the presence of the blue intercalating dye Hoechst or DAPI which is used to label nuclear DNA”. This statement appears incorrect for two reasons: 1. Hoechst is usually added to live cells in order to photosensitise and yield massive, readily detectable damage, not to just label DNA (if Hoechst were to be used as a label to mark DNA, it could be added after microirradiation); 2. Hoechst and DAPI are not DNA intercalators, they are rather minor groove binders (the mode of binding is complex and depends on a number of factors, including the type of DNA and a DNA/dye ratio).

A: We corrected and clarified that point. Actually using Hoechst helps identify the placement of the laser beam.
 
R: Minor comments:
It is unclear what the authors mean by referring to photoconversion of Hoechst, DAPI and VybrantDyeCycle and, in the same sentence and context, to blinking of YoYo described as “a change in optical properties”. What change of optical properties of YoYo do the authors have in mind?

A: In order to focus on Hoechst and DAPI we removed the comments on the other two dyes.
 
R: The authors state (in Discussion): “As an alternative nuclear marker, we suggest employing a fluorescently tagged protein that localizes at the nuclear periphery and does not interfere with the experimental process.”     Why use such a complicated approach? It is much simpler to detect a transmitted light image, preferably using phase contrast or Nomarski interference contrast, and mark the outline of the nucleus in image overlay.

A: We and other labs standardly use fluorescent labels of the nuclear envelope without complications. We are not sure transmitted light images are compatible with rapid time lapse imaging since filters have to be changed but we mentioned this possibility in the text.
 
R: The authors state: “However, there is evidence showing that DNA sensitization prior to laser exposure is not required:...” This is true, but again the way the authors put it is somewhat confusing and they refer to research in which UVA as well as visible light was used. Indeed, photosensitisation by exogenous DNA-binding compounds is never required – regardless of the type of light, UV or VIS. Moreover, adding a photosensitizer to the experimental system influences the type of damage. Thus, I suggest to distinguish two cases:

1. using UV-excited dyes and UV or near-UV (405nm) light, and
2. using dyes excited by visible light and visible light excitation.

 
In the case (1) the exciting light (UV or 405) will induce typical UV damage (PP, PD). Adding a photosensitizer prior to microirradiation will result in photodynamic effect, and cause induction of more types of lesions, and more extensive damage. There is vast literature about the action of UV alone and photodynamic effect type I and II. Some reference to this field of knowledge should be made in this paper.

A: We have added a section discussing the types of damage generated under different conditions.

R: Contrary to general belief, in the case (2) the exciting visible light alone, without any exogenous photosensitisers, will also induce DNA damage – single- and double-strand breaks, and recruitment of repair factors (Solarczyk et al., 2012, DNA Repair¹).

A: We noticed this phenomenon in our own experiments, and have now commented upon this in the text and have cited the reference suggested.

R: In summary, indeed various types of DNA damage can be induced without adding photosensitisers to cells prior to microirradiation, not only when UV, or 405 nm light is used, but also when visible light is applied. I suggest to clarify these facts in the paper.
 
 A few inaccurate statements should be straightened out:

• “A common approach used to assess DNA repair factor binding in mammalian cells is to induce DNA damage with a UV laser and follow the movement of GFP-tagged proteins to the site of damage”. To be precise, induction of (local) DNA damage by a focused laser beam is used to detect recruitment of repair factors, but not their binding per se.

A: True. Corrected.

• R: “Movement of GFP-tagged proteins to the site of damage”. Movement of GFP-tagged proteins is not detected (this can be done by FCS) – only local increase of a concentration of a fusion protein is detected, and this arises from recruitment.

A: Corrected.

R: The authors state:
„exposing DAPI or Hoechst to a strong UV laser” - light intensities and doses of energy delivered to the exposed region of the nucleus (area?) should be given.
Full information (dose) is lacking but it appears that the authors used an excessive power of 405nm light (12.8 mW at objective). DNA damage, especially in the presence of a photosensitizer, can be expected when using only microJ of energy (microW in the laser beam, seconds of exposure). This means that the photoconversion the authors describe most likely would have been less prominent, had a lower intensity and dose of energy been used. Applying excessive energy during microirradiation leads to such an extensive damage that relevant physiological studies may be impossible (Note that some images in the paper [Kong et al.] show microirradiation tracks in phase contrast images; the power which was used in this study was very high). I suggest that the authors state that the intensities (and doses of energy) they used may have been too high to induce DNA damage on the level encountered under typically encountered physiological conditions.

A: See response to Anna Fortuny, who raised this issue as well. Now we state that we use high laser power in order to demonstrate the effect and provide further information on the laser conditions. Furthermore, we refer to a study detecting and minimizing such an effect in their setup.
 
R: Typo:
“Here will illustrate the problem and suggest necessary controls.”  Here we will.

A: Corrected.
 
R: Hope this helps.

A: Yes. Thank you!

Thank you very much for your comments! Version 2 of this research note accommodates the changes that you suggested unless stated otherwise.

•  The authors refer to a “strong UV laser”. It is not clear which conditions are required for DAPI/Hoechst photoconversion (laser power, exposure time) and how they compare to classical exposure times for DAPI visualization and to UV laser settings for laser damage induction. It is important to clarify this point to determine if we face a marginal or a general problem. 

Indeed, we use higher laser power than used in most studies in order to demonstrate the photoconversion effect. The purpose of this research note is to increase the awareness of the phenomenon and highlight that a negative control is particularly essential in this experimental setup. Even small amounts of photoconverted dye can change image quantitation and may alter apparent recruitment dynamics. We do think that this effect is particularly relevant to the study of uncharacterized proteins. Explorative studies may tempt the researcher to vary laser power and exposure time because the degree of damage required for protein recruitment is not known. This bears the risk of a false positive result. A preventive measure is to generate the damage without using Hoechst.
We now cite a study in which photoconversion was detected and minimized, indicating the relevance of this note.
A recent report on photoconversion in standard multiple color microscopy applications specifies exposure times for photoconversion¹.

• The authors should be more specific when referring to UV laser or UV light. They should specify “UVA”, as other UV wavelengths such as UVC, also used to introduce local DNA damage (Dinant  et al.,2007¹), may not have the same effect.

We added a section on the type of damage generated +-sensitizing agents and cited Dinant et al.

• They could suggest using BrdU instead of Hoechst to pre-sensitize cells to UVA light as done in a number of studies (e.g. Lukas et al.,2003²). 

Now we mention BrdU as a sensitizing agent and specify which type of damage is generated in its presence (double strand breaks ²). However, we favor omission of sensitizing agents altogether.

• The authors illustrate the problem with GFP-tagged proteins but the issue would be similar with YFP-tagged proteins. They explain that far-red emission is compatible with photoconverted DAPI/Hoechst. How about red emission?

We have used GFP in our setup and therefore mainly discuss GFP. However, according to the publications we cited the effect may apply to YFP and other proteins with emission in the orange and near red as well ^1,3. This is now mentioned.

• There are 55 files on figshare, which seems excessive, and they are difficult to navigate through so in the end it is not very useful. 

We were asked to deposit the raw data on figshare. As mentioned in our note we also deposited avi files of timelapse MIPs in both channels as well as a channel merge. These files are are easy to handle.

• Result section: “cells expressing the GFP-tagged protein (Figure 1)”. These cells are not shown on the figure and which GFP-tagged protein is it?

We changed the position of the reference to Fig. 1 in this sentence to avoid implying that we will show cells expressing a GFP-tagged protein.
Rather than naming the protein we want to highlight that this type of false-positive result can apply to any GFP-tagged protein tested, which is not recruited to the damage site.
 
References
1) Karg, T. J. & Golic, K. G. Photoconversion of DAPI and Hoechst dyes to green and red-emitting forms after exposure to UV excitation. Chromosoma 127, 235-245, doi:10.1007/s00412-017-0654-5 (2018).
2) Ferrando-May, E. et al. Highlighting the DNA damage response with ultrashort laser pulses in the near infrared and kinetic modeling. Front Genet 4, 135, doi:10.3389/fgene.2013.00135 (2013).
3) Zurek-Biesiada, D., Kedracka-Krok, S. & Dobrucki, J. W. UV-activated conversion of Hoechst 33258, DAPI, and Vybrant DyeCycle fluorescent dyes into blue-excited, green-emitting protonated forms. Cytometry A 83, 441-451, doi:10.1002/cyto.a.22260 (2013).