RAD54L2 counters TOP2-DNA adducts to promote genome stability (Etoposide treated RPE1 CRISPR screens in TP53 and RAD53L2 knock outs)
Data files
Dec 06, 2023 version files 6.68 MB
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gattinara.counts.tsv
3.17 MB
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gattinara.symbols_ids.csv
938.99 KB
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gattinara.TP53_RAD53L2ko.drugz_output.tsv
1.12 MB
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gattinara.TP53ko.drugz_output.tsv
1.12 MB
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README.md
3.74 KB
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SPJ_DDR_v2.counts.tsv
194.06 KB
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SPJ_DDR_v2.symbols_ids.csv
43.18 KB
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SPJ_DDR_v2.TP53ko.drugz_table.tsv
49.26 KB
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SPJ_DDR_v2.WT.drugz_table.tsv
49.40 KB
Abstract
The catalytic cycle of topoisomerase 2 (TOP2) enzymes proceeds via a transient DNA double-strand break (DSB) intermediate termed the TOP2 cleavage complex (TOP2cc), in which the TOP2 protein is covalently bound to DNA. Anti-cancer agents such as etoposide operate by stabilising TOP2ccs, ultimately generating genotoxic TOP2-DNA protein crosslinks that require processing and repair. Here, we identify RAD54-like 2 (RAD54L2) as a factor promoting TOP2cc resolution. We demonstrate that RAD54L2 acts through a novel mechanism together with zinc finger protein associated with TDP2 and TOP2 (ZATT/ZNF451) and independent of tyrosyl-DNA phosphodiesterase 2 (TDP2). Our work suggests a model wherein RAD54L2 recognises sumoylated-TOP2 and, using its ATPase activity, promotes TOP2cc resolution and prevents DSB exposure. These findings suggest RAD54L2-mediated TOP2cc resolution as a potential mechanism for cancer-therapy resistance and highlight RAD54L2 as an attractive candidate for drug discovery.
https://doi.org/10.5061/dryad.fj6q57422
Counts and analyses of CRISPR screens.
Description of the data and file structure
File names prefixes indicate which library was used in the crispr screen:
- gattinara.* – Screens conducted with the Broad Gattinara library
- SPJ_DDR_v2.* – Screens conducted with the custom SPJ DDR v2 library (see doi.org/10.7554/eLife.55325)
And suffixes indicate the type of data:
- *.drugz_output.tsv – Results from analyses using DrugZ. Comparisons were of DMSO vs etoposide treated cells of the same genotype. See DrugZ paper for details of the algorithm and precise definitions of the statistics reported (https://doi.org/10.1186/s13073-019-0665-3). Columns defined below:
- GENE: Gene symbol, as used in the counts files.
- sumZ: Un-normalised summed Z-scores per gene.
- numObs: Number of observations (guides) per gene.
- normZ: Normalised summed Z-score.
- pval_synth: p-value of synthetic lethality (i.e. the combination of guides targeting the named gene and the treatment caused decreased abundance).
- rank_synth: Rank when ordered by most synthetically lethal.
- fdr_synth: False discovery rate corrected significance of pval_synth.
- pval_supp: p-value of synthetic viability (the opposite of synthetic lethality).
- rank_supp: Rank when ordered by most synthetically viable.
- fdr_supp: False discovery rate corrected significance of pval_supp.
- *.counts.tsv – CRISPR guide abundances
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The first two columns ("guide" and "gene") give CRISPR guide sequence and gene symbol for each row. Subsequent columns are guide abundances for the named sample.
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gattinara.counts.tsv sample details:
Replicate Treatment Days grown KO SPJas3042_RD54L2D10A 10 TP53|RAD54L2 SPJas3042_RD54L2D10B 10 TP53|RAD54L2 SPJas3042_RD54L2DMSA DMSO 25 TP53|RAD54L2 SPJas3042_RD54L2DMSB DMSO 25 TP53|RAD54L2 SPJas3042_RD54L2V16A Etoposide 25 TP53|RAD54L2 SPJas3042_RD54L2V16B Etoposide 25 TP53|RAD54L2 SPJas3042P53D10A 10 TP53 SPJas3042P53D10B 10 TP53 SPJas3042P53D25DMSOA DMSO 25 TP53 SPJas3042P53D25DMSOB DMSO 25 TP53 SPJas3042P53D25VP16A Etoposide 25 TP53 SPJas3042P53D25VP16B Etoposide 25 TP53 -
SPJ_DDR_V2.counts.tsv sample details (all grown 25 days):
Replicate Treatment KO 9_DMSO_D22 DMSO Wild-type 9_ETP_D22 Etoposide Wild-type 28_DMSO_D22 DMSO Wild-type 28_ETP_D22 Etoposide Wild-type c2_DMSO_D22 DMSO TP53 c2_ETP_D22 Etoposide TP53 c4_DMSO_D22 DMSO TP53 c4_ETP_D22 Etoposide TP53
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- *.symbol_ids.csv – Tables mapping symbols used in counts and DrugZ tables to more recent symbols, and various gene IDs. Columns defined below:
- Original_symbol: The symbol given in *.counts.tsv files.
- Symbol: Updated symbols, as used in the manuscript
- HGNC_ID, Ensembl_gene_ID, & NCBI_gene_ID: Database IDs for the named databases
CRISPR screens were performed as described in the manuscript. Comparisons of guide abundances in treated and untreated samples were made with DrugZ.
All comparisons are of DMSO vs Etoposide treated cells.
- D’Alessandro, Giuseppina; Morales-Juarez, David A.; Richards, Sean L. et al. (2023). RAD54L2 counters TOP2-DNA adducts to promote genome stability. Science Advances. https://doi.org/10.1126/sciadv.adl2108