DOI: 10.5061/dryad.fj6q57422
title: RAD54L2 counters TOP2-DNA adducts to promote genome stability (Etoposide treated RPE1 CRISPR screens in TP53 and RAD53L2 knock outs)

description: 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.
description: 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.
description: # CRISPR screen data 
 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
 (). 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 * 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. *
 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 | * *.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