The Protease WSS1A, the Endonuclease MUS81, and the Phosphodiesterase TDP1 Are Involved in Independent Pathways of DNA-protein Crosslink Repair in Plants

Abstract

DNA-protein crosslinks (DPCs) represent a severe threat to the genome integrity; however, the main mechanisms of DPC repair were only recently 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 homologs of the universal repair proteases SPARTAN/ weak suppressor of smt3 (Wss1), WSS1A, is essential for DPC repair in Arabidopsis (Arabidopsis thaliana). WSS1A defective lines exhibit developmental defects and are hypersensitive to camptothecin (CPT) and cis-platin. Interestingly, the CRISPR/Cas9 mutants of 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 METHYL METHANESULFONATE AND UV SENSITIVE PROTEIN 81 (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.

An Arabidopsis FANCJ helicase homologue is required for DNA crosslink repair and rDNA repeat stability

“…We further analysed the genetic interaction of FANCJB with the endonuclease MUS81. In Arabidopsis, MUS81 was postulated to define an important backup pathway for maintaining genome stability, as double mutants with a wide variety of DNA repair factors exhibit increased genotoxin sensitivity, severe growth retardation or synthetic lethality [31,37–39]. Our results clearly show that FANCJB is not processing CLs in a common pathway with MUS81 in plants.…”

“…As double mutants of MUS81 with the RTR complex partners RECQ4A and TOP3α or the FA helicase FANCM exhibit synthetic lethality, this further emphasizes the importance of these parallel pathways in the repair of replicative DNA damage [20,37,38]. MUS81 was only recently identified as the central player in DNA-protein crosslink repair, where the nuclease acts in parallel to the protease WSS1A and the tyrosyl-DNA-phosphodiesterase TDP1 [39]. REV3, the catalytic subunit of the translesion synthesis (TLS) DNA polymerase Zeta is an additional important player in ICL repair, as TLS acts downstream of the FA pathway and in the error-prone post-replicative repair [5].…”

The Aspartic Protease Ddi1 Contributes to DNA-Protein Crosslink Repair in Yeast

“…Albeit the protein moiety of a crosslink is the most evident target for repair, the existence of a proteasome-independent proteolytic pathway directed at DPCs was not addressed until recently (Duxin et al, 2014). This discovery was followed by the identification of the first DPC-dedicated proteases Wss1/SPRTN in different domains of life, including yeast, worms, mammals and plants (Enderle et al, 2019;Lopez-Mosqueda et al, 2016;Stingele et al, 2016;Stingele et al, 2014;Vaz et al, 2016). Up to date, the metalloproteases of the Wss1/SPRTN family remain the only enzymes which function is committed to DPC proteolysis.…”

Arabidopsis C‐terminal binding protein ANGUSTIFOLIA modulates transcriptional co‐regulation of MYB46 and WRKY33

“…S4b). Conversely, AN‐Myc expressed in the TDP1 T‐DNA insertional mutant tdp1‐2 (Enderle et al ., 2019) exhibited dramatically reduced association with MYB46 promoter in the ChIP analysis (Fig. 4a).…”