Peer review of: Real-time tracking reveals catalytic roles for the two DNA binding sites of Rad51 (2020)


Reviewers' comments: 

Reviewer #1 (Remarks to the Author): 

RAD51-driven DNA strand exchange is a prerequisite for homology-directed DNA repair. However, 
the detailed mechanism and dynamics of this process remain an enigma. To address this question, 
Ito et al. used fluorescence-based real-time tracking methods to characterize three DNA-binding 
related mutant variants of Rad51 (L1 and L2 in site I, and S2 in site II). The same group has 
previously shown that Rad51-driven DNA strand exchange involves two distinct three-strand 
intermediates, namely C1 and C2. Here, the authors found that (1) L1 (R257) is important for the 
formation of C1 intermediate by facilitating dsDNA capture; (2) L2 (V295) promotes C1-C2 
transition by stabilizing heteroduplex DNA in the C2 intermediate; and (3) S2 (R324 and K334) 
serves as an entry gate for both ssDNA and dsDNA during this process. Finally, a molecular 
simulation approach reveals the catalytic process of Rad51-mediated DNA strand exchange. The 
authors further propose that the insertion of the inter-triplet gap in the incoming duplex DNA by L2 
Val-295 leads a stable elongated nucleoprotein filament for the homology search. The novelty of 
this manuscript is appreciated and will be of interest to others in the wider community. However, 
the following concerns need to be addressed to strengthen this manuscript. 

Major concerns: 

1. The authors propose that stabilization of the elongated dsDNA filament requires a deep insertion
of L2 Val-295. It will be important to replace Val to Leu, instead of Ala, to verify this model.

2. The authors included the Swi5-Sfr1 auxiliary factor in various assays to see its effects on
Rad51. It will be important to verify whether these Rad51 variants are defective in the interaction
of Swi5-Sfr1.

3. Fig 1C-(b). Why is wild-type Rad51 ATPase activity not stimulated by ssDNA? It has been
documented that the Rad51 ATPase activity can be stimulated by ssDNA (Sauvageau et. al., 2005,
MCB). Consistent with this notion, why is S2 mutant defective in DNA binding but remains the
same ATPase activity as wild-type counterpart (comparing Fig 1C-(b) and Fig 4A)?

4. Fig 4C & 5C. It will be important to include a negative control to verify whether this FRET-based
assay could reflect the status of forming elongated presynaptic filaments by Rad51. One control
can be done by setting up the reaction without the presence of ATP.

5. Fig 4C & 5C. To rule out the effect that Rad51 dynamic assembly influences the elongation
between two FRET dyes, the authors should include AMP-PNP or Ca in the reaction to see whether
they could obtain the same results.

6. Fig 5C-(d). How could we evaluate the difference between wild-type and L2 variant is
meaningful? Appropriate controls or statistical analyses are required to make a strong argument.

7. A similar site II mutation has been examined in S. cerevisiae Rad51 (ScRad51). It has been
documented that ScRad51 containing mutations in site II (II3A; R188, K361, and K371) retains
the ability to form nucleoprotein filaments, but is fully defective in DNA strand exchange (Cloud et
al., 2012, Science). Note that ScRad51 K361 and K371 correspond to K324 and K334 in SpRad51.
The authors should compare and discuss their similarities and differences in the Discussion
session.

Specific points: 

1. Fig 3B-(c). This control experiment is not necessary to be included in the main figure. It should
be combined with Fig. S1.

 
Reviewer #2 (Remarks to the Author): 
 
Ito et al. analyze the role of two DNA binding sites in Schizosaccharomyces pombe Rad51 (site I 
and site II) in the catalysis of DNA strand exchange. The authors have used a variety of 
biochemical and biophysical approaches to examine the deficiencies in strand exchange activity of 
point mutants in the DNA binding motifs, including a mutant in each site I loop (L1 and L2) as well 
as a double mutant in site II (S2). Using fluorescence-based real-time assays, the authors show 
that the three mutants have distinct defects in processing the strand exchange intermediates 
(presynaptic filament, C1 (paranemic joint), and C2 (plectonemic joint)). 1) Rad51-S2 mutations 
abolish ss and dsDNA binding. Thus, the mutant is defective in both presynaptic filament formation 
and dsDNA capture, 2) Rad51-L1 mutant lacks dsDNA binding and is unable to form the C1 
intermediate. 3) Rad51-L2 is able to produce C1, but unable to transition to C2, as verified by the 
accumulation of C1 product in an abortive strand exchange assay. 
 
Taken together, the authors conclude that each of the examined components of the Rad51 binding 
sites have important but largely distinct roles in mediating Rad51-DNA interactions and transitions 
through the strand exchange process. This is a very nice study that complements what has been 
identified from the structural analyses of RecA and Rad51. It broadens our mechanistic 
understanding of DNA strand exchange carried out by general recombinases. 
 
There are a few concerns and suggestions that need to be addressed. 
 
Specific Comments 
1. The title of the manuscript is a too broad to suggest that the paper reveals more about strand 
exchange than it actually does. Consider swapping “process” for “details” to more appropriately 
describe the work and try to incorporate the fact that the data focus on the DNA binding domains. 
2. I recommend showing data for analysis of the three mutants (e.g., PAGE gel image, 
oligomerization status, and interaction with Swi5-Sfr1) somewhere in the manuscript. 
3. Fig. 1C and page 6: If the authors want to claim that the ATPase of three mutants are 
comparable to WT Rad51 in the strand exchange assay condition, they should include ATP 
hydrolysis data in the presence of both ss and dsDNA. Also, the authors didn't discuss the lower 
ATPase activity of L2. In conjunction with the mutants, can the authors address if (C1 to C2) 
transition or (C2 to ssDNA release) may require ATP binding/hydrolysis? 
4. Please explain in more detail how the amount of C1 % was calculated in Fig. 3A-f. 
5. While the FRET data in Fig. 4C support Rad51 filament formation, it is not clear if the lower 
FRET signal is due to “elongation” of the DNA as the authors suggest. If the authors can provide 
more evidence (e.g., the decrease of the FRET signal matches what they would expect from 1.5x 
B-form DNA), please do so. Otherwise, consider revising. Also, it would be interesting if they would 
measure FRET change during other transition, such as (C2 to ssDNA-release). 
6. Briefly explain why the S2 mutant should be a double-mutant. 
7. It would be intriguing to examine if the mutants show any distinct defect in cells. Do the 
mutants (L1 and L2) have an ability to protect replication fork, that primarily requires Rad51 
filament formation but not strand exchange activity? Discuss this issue at least. 
 
Minor Comments 
1. Page 12, paragraph 2; “not” should be “no.” 
2. Page 12, paragraph 3; “binging” should be “binding.” 
3. Page 19, the title of section that starts with “Analysis of association…” should lose the word 
“measuring.” 
4. Statistical significance to describe differences in activity would help support the author’s claims. 
For example, the authors suggest the FRET signal is different between Rad51 and Rad51-L2 in Fig. 
5C. Statistics would help here. 
5. Fig 1 A (b) and (c); It is difficult to identify the locations of the residues in the structure. I would 
recommend simplifying the figure and highlight the residues. 

 
 
 
Reviewer #3 (Remarks to the Author): 
 
The manuscript by Ito et al uses a combination of real-time ensemble FRET and 2AP substitutions 
to reveal important findings regarding how Rad51 filaments promote DNA strand exchange. 
Although the presence of several three-strand intermediate states along the exchange pathway 
has already been demonstrated, this works provides the first unambiguous insight into how DNA 
binding sites I and II distinctively impact the formation of C1 and C2 intermediates. Using specific 
mutations, the work clarifies the role of L1 and L2 loops in Site I at different stages of the process 
and suggests a previously unknown role for Site II as an entry gate not only for dsDNA but also for 
ssDNA. 
The manuscript is concise, well written and highlights the key findings. The work makes a key 
contribution to the field as it is likely that other RecA-like recombinases will function through a 
similar mechanism. 
I support its publication after the authors have addressed the comments below: 
1. Figure 2A-b and 2B-b show the time-dependent variation in fluorescein signal upon addition of 
WT and several mutants using the FRET pairing and displacement assay, respectively. Given that 
they have a FRET essay, I would like to have seen the simultaneous recording of both D and A, or 
alternatively, at least a control with no Rhodamine present. It is known that the addition of 
proteins might quench or enhanced fluorescence and in the absence of these controls whether the 
changes in signal over time are due exclusively to FRET is not demonstrated 
2. It will have been useful if instead of relative fluorescence in Figure 2Ab and Figure 2Bb, the 
authors transformed this relative change into the amount of product formed. This will allow us to 
assess the DNA strand exchange efficiency monitored by these assays and compare it with 
literature values or with gel-based assays. As it is and without knowing the relative intensity of the 
D in the fully exchanged DNA strand it is not possible to know what is the level of final product 
formed. 
3. Table S2 lists the values of rate constants obtained followed the scheme in Figure 2C and these 
are plotted in panels 2Ca-f. The authors mentioned that these values have been obtained by 
‘simulating’ each reaction (pairing and displacement) using the DynaFit software. How well the 
simulated curve fits the experimental curve is not shown, this should be graphically included in the 
supplementary section for each curve fitted using DynaFit. 
4. Figure 2A-b shows a biphasic variation in the time-dependent signal for the WT and L2 mutants 
with and without S5S12. What is the mechanism behind this biphasic variation? The biphasic 
change does not seem to be present when following the pairing assays using 2AP 
5. In contrast to most EDTA-trapping experiments, Figure 2Ac shows at each injection point a 
much lower intensity than the threshold trace, what is causing this? 
 
Minor points. 
In most schematics, the D and A ‘stars’ are not easy to differentiate 
In figure 2Af, it is difficult to distinguish the colors of the different WT and variants 
Figure 5Cd please label the gel bands 
In Figure 4c and 5Cb-c the Y-axis for the spectra plots reads ‘Normalized int.’ It is unclear to me in 
which way it has been normalized, please clarify