Peer review of: BRCA1-associated structural variations are a consequence of polymerase theta-mediated (2020)


 
Reviewer #1 (Remarks to the Author): 
 
In this investigation, Kamp et al. report that C. elegans lacking the mammalian BRCA1 and BARD1 
orthologs accumulate mutations similar to those seen in human brca1-deficient tumors. These 
mutations include relative small (<30 bp) deletions and large (1-1000kb) tandem duplications, both of 
which have microhomologous sequences at the breakpoints and/or small insertions. In addition, an 
increase in base substitutions is observed. They also show that polymerase theta mediated end 
joining, but not classical non-homologous end joining, is responsible for the majority of the deletions 
and tandem duplications. 
 
While the role of POLQ in promoting deletions with accompanying microhomology is well-established, 
its involvement in the formation of tandem duplications has not been reported. This is the novel 
aspect of this paper. The authors propose that the tandem duplications could result from aborted 
homologous recombination, when one strand invades into a homologous template and DNA synthesis 
occurs, but the nascent strand is unable to anneal with the other broken end. They argue that this 
would be more likely in the absence of BRC-1 and BRD-1, due to a defect in resection. 
 
My major concern is that while this model is attractive, the authors provide no direct support. They do 
cite other analyses showing that tandem duplications are common in human tumors with brca1 
mutations, but not with brca2 mutations, but this is only correlative data. Because the main novelty of 
this study lies in the proposed mechanism of tandem duplication formation, some experimental 
support of their model is needed. The most obvious prediction, that a brc-2 mutation would prevent 
their formation, cannot be tested due to the necessity of brc-2 for meiotic recombination. Perhaps the 
authors can look in an mre11 brc-1 double mutant (although these have reduced fertility)—the 
prediction would be that tandem duplications should also be more frequent due to a defect in 
resection. Alternatively, a mutation or condition that increases the amount of resection at double-
strand breaks should decrease the frequency of tandem duplications. The inclusion of additional data 
to elucidate the conditions which allow TMEJ-mediated tandem duplication formation would 
significantly strengthen the manuscript. 
 
Other points: 
 
1. The Scully lab proposed an alternative model for tandem duplication formation involving replication 
restart-bypass in brca1 mutant cells (Willis et al., 2017, PMID 29168504). While the current 
manuscript does reference this work, a more through discussion of this model is warranted. 
 
2. The model for TMEJ repair following aborted HR is not novel. This was suggested at least two 
previous publications: Wyatt et al., 2016 (PMID 27453047) and Chan et al., 2010 (PMID 20617203). 
These papers should be cited when the model is proposed. 
 
3. A synergism between HR and TMEJ in the repair of IR-induced damage was previously reported in 
Chan et al., 2010. In this instance, mutation of both RAD51 and POLQ resulted in hypersensitivity to 
IR, similar to that reported in the current study. This should be cited. 
 
4. Lines 98-99: More detail about the type of somatic cells and how HDR was monitored should be 
provided here, so the readers don’t have to rely only on Figure S1. 
 
5. Line 103: Are the accompanying insertions templated from flanking sequences, as has been 
reported for other TMEJ junctions? 
 
6. Line 110: I seem to recall that earlier papers from this group showed that deletions that occur at 
G4 sequences almost always involved 1 nt of microhomology. Is the proportion different in brc-1 
mutants and if so, why might this be? 

 
7. Lines 174-175: this statement is oversimplified, as it is true only for relatively small deletions (Fig. 
3). 
 
8. Lines 189-198: This paragraph is confusing as written. Are the authors arguing that TMEJ is more 
common in C. elegans than in mammals? The phrase “provides an unlikely explanation” makes this 
unclear. 
 
9. Line 263: The authors claim that the reliance on short microhomologies sets TMEJ apart from MMEJ, 
but there are no consistent guidelines for MMEJ in the literature-- particularly as it is defined 
differently in yeast vs. other organisms, as the next sentence points out. MMEJ is often used as a 
general term for the use of any amount of microhomology during break repair. Therefore, it would be 
better to use different terminology here. 
 
 
Reviewer #2 (Remarks to the Author): 
 
The work of Kamp et al. proposes a key role for the DNA polymerase theta in generating mutation 
patterns that are known to be characteristic of homologous recombination (HR)-deficient human cells, 
using a worm germline mutagenesis model. These are important findings because they shed light on 
the mechanisms underlying mutagenesis under HR deficiency. An implication of these results is that 
they further suggest relevance of therapeutically targeting DNA polymerase theta in those HR-
deficient tumors that exhibit certain mutation patterns. Overall the methodology appears quite solid 
and the presentation balanced. I do have a few queries that need to be addressed. 
 
1. The conclusions have some potential to be affected by technical issues stemming from mutation 
calling from short reads, which is error-prone in case of calling short indels and moreso in case of 
larger structural variants. 
- only a single tool to call structural variants is used (Pindel); more modern tools (e.g. Manta, Gridss 
etc) may give more accurate results, e.g. in terms of placing the breakpoint, or in terms of false 
negative rates; note that the latter cannot be prevented by manual inspection of calls which they have 
done. Running an additional caller would help ensure the results are not skewed towards biases of a 
single algorithm. In particular different tools may have different biases with respect to lengths/types 
of variants they can detect, which can be relevant for the statements such as line 117 "The rate of 
TDs in brc-1 and brd-1 mutants is approximately tenfold lower than the rate of deletions in these 
mutants." 
- would the conclusions be affected if limiting to variant calls with higher confidence scores and/or in 
less difficult to align (less repetitive) genome regions? 
- please state average and/or median depth of sequencing in the methods. 
 
2. Data presentation issues. 
- there is much overplotting of the dots in 1A/1B/2A/2B/3A/3B which makes it hard to judge the 
density of points of different colors (microhomology lengths), affecting interpretation of results. I 
suggest to sort the points (x axis) by # nucleotides of microhomology, within each genotype. 
- line 142 "we observed a small increase in deletion mutagenesis in cku-80 and lig-4" and line 146 
"base substitution  rate is mildly increased in cku-80 and lig-4 mutants as compared to wildtype". I 
would not say this is a small nor mild increase (>=2 fold in Figs 2C/2E). In 2E, the SNV rate increase 
from NHEJ  KOs is bigger than for brc-1 KO. Consider rephrasing. 
- please draw two-sided error bars on bar charts 
- mutation rates flanking the breaks do not appear to be elevated, arguing against common use of an 
error-prone polymerase to extend past pol theta activity. Pol theta itself does not appear to contribute 
many SNVs according to Fig 3e. Thus currently the cause of higher SNV mutation rates in brc-1/brd-1 
backgrounds is unclear. Would looking at the trinucleotide mutation spectrum, and examining 
correlations to known mutation signatures from human tumors, be helpful in elucidating this? 
 

3. Multiple lines of animals k.o. for various genes were propagated, and mutation data pooled together 
for analysis. Are the results overall consistent between the independant lines? Potentially some of the 
observations might be driven by an acquired mutator in one of the lines (e.g. the increased SNV rate 
in NHEJ deficient lines). 
 
4. Regarding negative selection. Lines 179-181 "Deletions of that size will affect multiple genes in a 
gene-dense organism suchas C. elegans and their presence are likely counterselected for in animal 
propagation experiments". It is well possible that many of the smaller deletions are also under 
selection. Please discuss and/or address with analyses if this has the potential to bias results (e.g. 
there might be confounding between chromosomal locations of fitness genes and the chromosomal 
location bias of activity of the various HR failure-related mutational processes). 
 
5. A 1 nt microhomology has a rather high probability of occuring at random (depending on the local 
nucleotide composition). On the one hand, this affects interpretation of the data shown in plots, where 
1 nt microhomologies, often arising at random, are lumped together with 2 and 3 nt microhomologies, 
which are more likely to stem from real signal. On the other hand, this can affect interpretation of 
statements such as line 110: "These deletions are also characterized by an overrepresentation of 
micro-homology: 58% of deletions had at least one nucleotide that could be mapped to either 
junction". A baseline estimate of microhomology should be provided, if relying on 1 nt matches to call 
MHs. 
 
Minor. 
- typos: "undistinguisable", "we have here describe (~10 kb)". Line 169 might need to refer to Fig 3A. 
- line 174: "Our data demonstrate that all deletions with ≤6 nt of homology are the result of 
polymerase theta on DSBs". This is correct, however if I understand correctly this 6nt threshold might 
be better placed at a higher number of nucleotides, depending on the lengths of the microhomology 
segments in polq-1 mutants in Fig 3A, which are not evident from the figure. 
- line 107: "We found no significant difference in deletion rate between brc-1 and brd-1 (p=0.236), 
arguing against a BRC-1-independent role for BRD-1 in deletion prevention, and vice versa". I would 
not say this is necessarily very strong evidence, in the absence of experiments on the brc-1 brd-1 
double mutant, which they did not perform.