RAD54L2 counters TOP2-DNA adducts to promote genome stability.

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. Anticancer agents such as etoposide operate by stabilizing TOP2ccs, ultimately generating genotoxic TOP2-DNA protein cross-links 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 tyrosyl-DNA phosphodiesterase 2 (TDP2) and TOP2 (ZATT/ZNF451) and independent of TDP2. Our work suggests a model wherein RAD54L2 recognizes 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.

Parallel CRISPR-Cas9 screens clarify impacts of p53 on screen performance.

CRISPR-Cas9 genome engineering has revolutionised high-throughput functional genomic screens. However, recent work has raised concerns regarding the performance of CRISPR-Cas9 screens using TP53 wild-type human cells due to a p53-mediated DNA damage response (DDR) limiting the efficiency of generating viable edited cells. To directly assess the impact of cellular p53 status on CRISPR-Cas9 screen performance, we carried out parallel CRISPR-Cas9 screens in wild-type and TP53 knockout human retinal pigment epithelial cells using a focused dual guide RNA library targeting 852 DDR-associated genes. Our work demonstrates that although functional p53 status negatively affects identification of significantly depleted genes, optimal screen design can nevertheless enable robust screen performance. Through analysis of our own and published screen data, we highlight key factors for successful screens in both wild-type and p53-deficient cells.

The invention of CRISPR-Cas9 genome editing has unlocked a greater understanding of the human genome. Researchers can use this system to make targeted cuts in any gene in the genome, forcing the cell to perform a rapid repair at the cut site. These repairs often introduce mutations into the damaged area, adding or removing DNA letters and disrupting the gene. This allows researchers to study what happens to cells when specific genes are missing, which can help to uncover what each gene is for. One of the most comprehensive ways to use this technique is to perform a CRISPR-Cas9 screen, which disrupts each gene in the genome one by one. For a CRISPR-Cas9 screen to work well, a cell needs to survive the cuts to its genome. But there is a crucial gene that can stop this happening. Often described as the 'guardian of the genome', this gene codes for a protein called p53, a tumour suppressor that helps to stop a cell turning cancerous when its DNA becomes damaged. This protein activates when the cell senses a cut in its genetic material and can kill the cell if it fails to make a successful repair. Recent work has shown that the presence of a working copy of the gene for the p53 protein might limit the ability of CRISPR-Cas9 to edit genes. But the evidence was inconclusive. So, Bowden, Morales-Juarez et al. performed two parallel CRISPR-Cas9 screens in human cells with and without p53 to find out more. This revealed that CRISPR-Cas9 can inactivate genes in both normal cells and cells lacking the p53 protein, but that it works better in cells without p53. This was because, when p53 was active, the cells initiated a protective response against the CRISPR-Cas9 cuts. This changed the patterns of genes successfully inactivated by the screen, but it did not make the results unusable. Careful experimental design and thorough data analysis made it possible to get useful results even in cells with functional p53 protein. The gene for p53 has mutations in around half of human cancers. So, understanding how it affects CRISPR-Cas9 screens could influence the design of future experiments. It is possible that the effects of the p53 protein could vary from cell type to cell type, and with different p53 mutations. Comparisons like the one performed here could help to further unpick how the cell's DNA repair systems might interfere with future CRISPR experiments.