BRCA1-associated structural variations are a consequence of polymerase theta-mediated end-joining

Failure to preserve the integrity of the genome is a hallmark of cancer. Recent studies have revealed that loss of the capacity to repair DNA breaks via homologous recombination (HR) results in a mutational profile termed BRCAness. The enzymatic activity that repairs HR substrates in BRCA-deficient conditions to produce this profile is currently unknown. We here show that the mutational landscape of BRCA1 deficiency in C. elegans closely resembles that of BRCA1-deficient tumours. We identify polymerase theta-mediated end-joining (TMEJ) to be responsible: knocking out polq-1 suppresses the accumulation of deletions and tandem duplications in brc-1 and brd-1 animals. We find no additional back-up repair in HR and TMEJ compromised animals; non-homologous end-joining does not affect BRCAness. The notion that TMEJ acts as an alternative to HR, promoting the genome alteration of HR-deficient cells, supports the idea that polymerase theta is a promising therapeutic target for HR-deficient tumours.


Statistics
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A description of all covariates tested A description of any assumptions or corrections, such as tests of normality and adjustment for multiple comparisons A full description of the statistical parameters including central tendency (e.g. means) or other basic estimates (e.g. regression coefficient) AND variation (e.g. standard deviation) or associated estimates of uncertainty (e.g. confidence intervals) For null hypothesis testing, the test statistic (e.g. F, t, r) with confidence intervals, effect sizes, degrees of freedom and P value noted

Software and code
Policy information about availability of computer code Data collection Image analysis, base calling and error calibration were performed using standard Illumina software: BCL output from the HiSeqX and Novaseq6000 platform was converted using bcl2fastq tool (Illumina, versions 2.17 to 2.20 have been used) using default parameters. Raw reads were mapped to the C. elegans reference genome (Wormbase release 235) by BWA (version 0.7.17) and further processed via SAMtools(1.10).

Data analysis
Pindel (0.2.5a8), GATK(4.0.8.1), Manta(1.6.0) and GRIDSS(2.8.0) were used for callling structural variations, GATK(4.0.8.1) was used for SNV calling. Mutations were visually inspected using IGV (2.8.3) For manuscripts utilizing custom algorithms or software that are central to the research but not yet described in published literature, software must be made available to editors and reviewers. We strongly encourage code deposition in a community repository (e.g. GitHub). See the Nature Research guidelines for submitting code & software for further information.

Data
Policy information about availability of data All manuscripts must include a data availability statement. This statement should provide the following information, where applicable: -Accession codes, unique identifiers, or web links for publicly available datasets -A list of figures that have associated raw data -A description of any restrictions on data availability Raw sequences have been made publicly available at NCBI SRA (Accession code PRJNA599297). Data for N2 wild-type and polq-1 animals were published previously and can be found at NCBI SRA (Accession codes PRJNA260487 and PRJNA196232). A list of all events and raw data of figure 4 are available as supplementary data. No unreported custom computer code was used during this study. Database used: C. elegans reference genome (Wormbase -release 235).

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Life sciences study design
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Sample size
For NGS experiments we found in previous publications that ~3*50 generations/genotype provides a sample size sufficient to analyze in case mutants have a different mutation signature (see Data exclusions No data was excluded.

Replication
Apart from the usual performing experiments in multiplo, if possible, we used both brc-1 and brd-1 alleles in the experiments, which functionally require each other, to ensure reproducibility. No differences were observed, as depicted in figure 1 in the manuscript. We also performed the IR experiment with brd-1 and brd-1 polq-1 strains instead of brc-1 and brc-1 polq-1 and we obtained similar results. We also performed an IR experiment with another brc-1 polq-1 strain, in which the brc-1 allele was another null allele than used in this study. Again, we found similar results.
Randomization Allocation was random: For each experiment, the first worm (in the right developmental stage for the experiment) that was seen through the microscope when the stock plate was observed was picked precluding potential bias introduced by the experimentator Blinding Some experiments were blinded: the genotype and dose (if applicable) was not known when scoring the plates/microscopy slides in the IR experiment (fig 4), HDR reporter experiment ( fig S1) and RAD-51 staining ( fig S2). The mutation accumulation line experiment was not blinded, since the events were called by software and not scored by humans.