The protease WSS1A, the endonuclease MUS81 and the phosphodiesterase TDP1 are involved in independent pathways of DNA-protein crosslink repair in plants.

Although DNA-protein crosslinks (DPCs) represent a severe threat to the genome integrity, only recently main mechanisms of DPC repair were elucidated in humans and yeast. Here we define the pathways for DPC repair in plants. Using CRISPR/Cas9, we could show that only one of two homologues of the universal repair proteases SPRTN/WSS1 (WSS1A) is essential for DPC repair in Arabidopsis. WSS1A defective lines exhibit developmental defects and are hypersensitive to camptothecin (CPT) and cis-platin. Interestingly, the CRISPR/Cas9 mutants of the tyrosyl-DNA phosphodiesterase 1 (TDP1) are insensitive to CPT, and only the wss1A tdp1 double mutant reveals a higher sensitivity than the wss1A single mutant. This indicates that TDP1 defines a minor backup pathway in the repair of DPCs. Moreover, we found that knock out of the endonuclease MUS81 results in a strong sensitivity to DPC-inducing agents. The fact that wss1A mus81 and tdp1 mus81 double mutants exhibit growth defects and an increase in dead cells in root meristems after CPT treatment, demonstrates that there are three independent pathways for DPC repair in Arabidopsis. These pathways are defined by their different biochemical specificities, as main actors, the DNA endonuclease MUS81 and the protease WSS1A, and the phosphodiesterase TDP1 as backup.