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\begin{document}

\title{PREreview of bioRxiv preprint ``A Role for RNA and DNA:RNA hybrids in
the modulation of DNA repair by homologous recombination.''}



\author[1]{Tovah Day }%
\author[1]{Ramya Parasuram}%
\author[1]{Tasmia}%
\author[1]{Hongyun Qi}%
\author[1]{brendan_price}%
\author[2]{Nealia House}%
\author[2]{ozge_yuzugullu}%
\affil[1]{PREreview}%
\affil[2]{Affiliation not available}%


\vspace{-1em}



  
  \date{May 25, 2024}


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\textbf{PREreview}

A role for RNA and DNA:RNA hybrids in the modulation of DNA repair by
homologous recombination

https://doi.org/10.1101/255976

~

\textbf{Summary}

~

D'Alessandro \emph{et al.} proposes mechanistic advances for the role of
damage-induced long-non-coding RNAs (dilncRNAs) in double-strand break
(DSB) repair.~ The authors have shown that DNA:RNA hybrids can
accumulate at targeted DSBs in transcribed and non-transcribed regions.~
In addition, damage-induced hybrids accumulate predominantly in S/G2 and
are promoted by DSB resection.~ Although inhibition of transcription did
not reduce resection, the authors reported that it reduced recruitment
of HR proteins.~ In perhaps their most compelling result, the authors
demonstrate that sequence-specific inhibition of dilncRNAs corresponding
to a targeted break result in impaired homologous recombination.~
Finally, the authors find evidence that RNase H2A is recruited to DSBs
in a complex with BRCA2 and interestingly, BRCA2 depletion leads to
accumulation of DNA:RNA hybrids at DSBs.~ These results lead them to
suggest a novel mechanism in which DNA:RNA hybrids promote recruitment
of HR factors and RNase H2 in the early stages of repair, followed by
degradation of the DNA:RNA hybrid by RNaseH2 and repair of the break via
HR.

~

\textbf{Positive Feedback}

~

We found the results showing that DR-GFP signal goes down with ASOs
around the breaksite to be both surprising and exciting.~ It was useful
to learn that ASOs can be used to specifically inhibit nuclear RNAs
which is distinct from inhibiting cytoplasmic RNAs.~ In addition, the
dual observations that BRCA2 recruits RNase H2A and siBRCA2 increases
hybrid accumulation provide nice support for the authors' model
regarding removal of hybrids at late stages of repair.~

~

~~~~~~~~~~~ The study led us to many questions and future exciting
directions this research could take including:

~

1.~~~~ If you only block hybrids on one side of the breaksite, do you
get the same reduction in HR?

2.~~~~ Does chewing the hybrid up result in a different outcome than
unwinding?~ Why does BRCA2 bring RNase H2A instead of bringing
Senataxin?

3.~~~~ Does this mean that BRCA-null tumors have accumulations of
DNA:RNA hybrids at DSBs?

4.~~~~ BRCA1 and BRCA2 have roles in replication as well -- do they also
recruit RNase H2A to replication forks by a similar mechanism?

~

~

\textbf{Major Concerns}

\textbf{~}

For the results in Figure 1, it is important to formally demonstrate
that the non-genic regions are not being transcribed to claim \emph{de
novo} recruitment of RNA Pol II.~ We thought that RNA-seq would be an
appropriate control to show this in your system.

~

We were concerned that the results presented do not fully support the
following statement:

~

``These results show that dilncRNAs, while not affecting DNA-end
resection, contribute to the recruitment of HR proteins to the site of
damage and, by doing so, they promote HR.''

~

We felt this statement was an over-interpretation of the data and needs
to be softened or supported with more data.~ The data show that
\selectlanguage{greek}α-\selectlanguage{english}amanitin and DRB treatment lead to less recruitment of HR factors and
ASOs lead to less DR-GFP signal. ~\selectlanguage{greek}α-\selectlanguage{english}amanitin and DRB lead to massive
transcriptional profile changes in the cell, thus it is difficult to
extrapolate from their effects.~ In addition, \selectlanguage{greek}α-\selectlanguage{english}amanitin and DRB are
also two steps removed from the existence of the dilncRNAs at the
breaksite.~ However, the model would be greatly strengthened by
experiments that combine site-specific ChIP at the I-Sce1 break with
ASOs showing reductions in recruitment of BRCA1, BRCA2, and RAD51.

~

We felt the italicized text below represented an assertion that was too
strong based on the data,

~

``\emph{Based on our observations that dilncRNAs form DNA:RNA hybrids in
S/G2-phase cells and control the recruitment of HR proteins to sites of
DNA damage}, we sought to test the involvement of DNA:RNA hybrids in the
focal accumulation of HR proteins at DSBs.''

~

Focal accumulation of HR proteins was attenuated but clearly still
occurs.

~

The authors need to show that the ASOs are reducing the abundance of the
I-Sce1-induced lncRNAs in the experiments of Figure 3e and f; this was
an important missing control.

~

In Figure 4, the stoichiometry of the \emph{in vitro} binding is
important for the proposed mechanism. Also, a negative control is
important to assert specificity of binding.

~

In Figure 6, if ``sorting of the S/G2-phase cells population was
necessary since BRCA2 inactivation affects the cell cycle (data not
shown)'' is true for the experiments in 6d, then it must also be true
for the experiments in 6a which appeared to be done in asynchronous
cells.

~

Finally, we felt that the overall model would be strengthened by more
discussion of the authors' previous recent paper, Michelini \emph{et
al.}, 2017.~ For example, how do DDRNAs fit into this model?~ In
particular, how does their role differ from the DNA:RNA hybrid?

\textbf{~}

~

\textbf{Minor Concerns}

\textbf{~}

1.~~~~ In Fig 1c, it would be more convincing to include a negative
control (without I-PpoI) to show the background level.

2.~~~~ In Fig 2b, the zoomed fields do not match the squared areas in
the upper image.

3.~~~~ In Fig 2c and 4b, we found the y-axis metric, ``Normalized number
of yH2A.X-DNA-RNA hybrids overlaps relative to random'' confusing. ~It
would be clearer the calculation of this metric was included in the
figure legend.

4.~~~~ In Supplementary Figure 3h, G1 and G2 fractions should be
different colors.

5.~~~~ Figure 5a and b -- should indicate how far away from the
breaksite the primers are and what the `unrelated' site is (either in
the figure or legends).

6.~~~~ It would be less confusing to represent Figure 6c as two
different IPs of RNase H2 rather than have two panels of RNase H2A in
the same columns.

7.~~~~ In Fig 6e, annotation for the blue Pac-Man is missing. Is it
CtIP? Also, as pictured, the interaction of RPA and DNA:RNA hybrids is
confusing (is RPA directly binding the hybrids?)~ We would consider
excluding RPA from the model since there is little evidence for when and
how RPA is interacting with the DNA:RNA hybrids.

~

~

~

~

~

~

\textbf{~}

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