SMC5/6 complex-mediated SUMOylation stimulates DNA–protein cross-link repair in Arabidopsis

Abstract

DNA-protein crosslinks (DPCs) are highly toxic DNA lesions consisting of proteins covalently attached to chromosomal DNA. Unrepaired DPCs physically block DNA replication and transcription. Three DPC repair pathways have been identified in Arabidopsis (Arabidopsis thaliana) to date: the endonucleolytic cleavage of DNA by the structure-specific endonuclease MUS81; proteolytic degradation of the crosslinked protein by the metalloprotease WSS1A; and cleavage of the crosslink phosphodiester bonds by the tyrosyl phosphodiesterases TDP1 and TDP2. Here we describe the evolutionary conserved STRUCTURAL MAINTENANCE OF CHROMOSOMEs SMC5/6 complex as a crucial component involved in DPC repair. We identified multiple alleles of the SMC5/6 complex core subunit gene SMC6B via a forward-directed genetic screen designed to identify the factors involved in the repair of DPCs induced by the cytidine analog zebularine. We monitored plant growth and cell death in response to DPC-inducing chemicals, which revealed that the SMC5/6 complex is essential for the repair of several types of DPCs. Genetic interaction and sensitivity assays showed that the SMC5/6 complex works in parallel to the endonucleolytic and proteolytic pathways. The repair of zebularine-induced DPCs was associated with SMC5/6-dependent SUMOylation of the damage sites. Thus, we present the SMC5/6 complex as an important factor in plant DPC repair.

The interplay of homology‐directed repair pathways in the repair of zebularine‐induced DNA–protein crosslinks in Arabidopsis

“…This connects HR and MMEJ pathways with the repair of MET1-DPCs. Moreover, we show that although the role of SMC5/6 is mainly connected with its function in HR (Diaz et al, 2019;Watanabe et al, 2009), it also fulfills other functions in DPC repair, most likely through its SUMO E3 ligase activity (Dvo r ak Toma st ıkov a, Prochazkova, et al, 2023;Yang et al, 2021). We identified three HZE2/RTEL1 missense variants, hze2-3, hze2-4, and hze2-5, with mutations in the ATPbinding pocket of the helicase domain, which belongs to the highly conserved SF2 helicase family.…”

“…Therefore, we conclude that RTEL1 and TEB function in parallel and synergistically during seed development, possibly by protecting the genome against replicative stress. To explore functional relationships of RTEL1/HZE2, TEB/HZE3, and previously characterized SMC6B/HZE1 factor (Dvo r ak Toma st ıkov a, Prochazkova, et al, 2023), we generated rtel1-1 smc6b-1 and teb-1 smc6b-1 homozygous double mutants.…”

“…The HZE genetic screen was performed as described (Dvo r ak Toma st ıkov a, Prochazkova, et al, 2023). Briefly, the W35 reporter line (Willing et al, 2016) was mutagenized with 0.2% ethyl methanesulfonate.…”

“…The first complementation group, HZE1 , was mapped to the STRUCTURAL MAINTENANCE OF CHROMOSOME 6B ( SMC6B ) subunit of the SMC5/6 complex. We showed that the SMC5/6 works in parallel to known DPC repair pathways, and its SUMOylation activity plays a critical role (Dvořák Tomaštíková, Prochazkova, et al., 2023). Here, we describe two new mapped genes HZE2 as RTEL1 and HZE3 as TEB , in which RTEL1 and TEB are mutated.…”

Revealing the transitory and local effect of zebularine on development and on proteome dynamics of Salix purpurea

“…In this concern, reduced root growth was observed in Arabidopsis in response to zebularine and in association with DNA damage and the activation of DNA repair pathways. Specifically, zebularine-induced DNA damage is associated with the formation of cross-links between MET1 and DNA during replication ( Nowicka et al., 2020 ; Prochazkova et al., 2022 ; Dvořák Tomaštíková et al., 2023 ). On the other hand, the available literature highlights the efficiency of zebularine in inducing homogeneous and effective DNA hypomethylation with few experimental artifacts caused by cytotoxicity and a relatively long half-life compared with azacytidine ( Cheng et al., 2003 ; Baubec et al., 2009 ).…”