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DNA-protein crosslinks (DPCs) are highly toxic DNA lesions represented by proteins covalently bound to the DNA. Persisting DPCs interfere with fundamental genetic processes such as DNA replication and transcription. Cytidine analog zebularine (ZEB) has been shown to crosslink DNA METHYLTRANSFERASE1 (MET1). Recently, we uncovered a critical role of the SMC5/6-mediated SUMOylation in the repair of DPCs. In an ongoing genetic screen, we identified two additional candidates, HYPERSENSITIVE TO ZEBULARINE 2 and 3, that were mapped to REGULATOR OF TELOMERE ELONGATION 1 (RTEL1) and polymerase TEBICHI (TEB), respectively. By monitoring the growth of hze2 and hze3 plants in response to zebularine, we show the importance of homologous recombination (HR) factor RTEL1 and microhomology-mediated end-joining (MMEJ) polymerase TEB in the repair of MET1-DPCs. Moreover, genetic interaction and sensitivity assays showed the interdependency of SMC5/6 complex, HR, and MMEJ in the homology-directed repair of MET1-DPCs in Arabidopsis. Altogether, we provide evidence that MET1-DPC repair in plants is more complex than originally expected., (© 2024 The Author(s). The Plant Journal published by Society for Experimental Biology and John Wiley & Sons Ltd.)

Structural maintenance of chromosomes (SMC) complexes are molecular machines ensuring chromatin organization at higher levels. They play direct roles in cohesion, condensation, replication, transcription, and DNA repair. Their cores are composed of long-armed SMC, kleisin, and kleisin-associated subunits. Additional factors, like NSE6 within SMC5/6, bind to SMC core complexes and regulate their activities. In the human HsNSE6/SLF2, we recently identified a new CANIN domain. Here we tracked down its sequence homology to lower plants, selected the bryophyte Physcomitrium patens, and analyzed PpNSE6 protein-protein interactions to explore its conservation in detail. We identified a previously unrecognized core sequence motif conserved from yeasts to humans within the NSE6 CANIN domain. This motif mediates the interaction between NSE6 and its NSE5 partner in yeasts and plants. In addition, the CANIN domain and its preceding PpNSE6 sequences bind both PpSMC5 and PpSMC6 arms. Interestingly, we mapped the PpNSE6-binding site at the PpSMC5 arm right next to the PpNSE2-binding surface. The position of NSE6 at SMC arms suggests its role in the regulation of SMC5/6 dynamics. Consistent with the regulatory role of NSE6 subunits, Ppnse6 mutant lines were viable and sensitive to the DNA-damaging drug bleomycin and lost a large portion of rDNA copies. These moss mutants also exhibited reduced growth and developmental aberrations. Altogether, our data showed the conserved function of the NSE6 subunit and architecture of the SMC5/6 complex across species., (© 2023 The Authors. The Plant Journal published by Society for Experimental Biology and John Wiley & Sons Ltd.)

DNA-protein cross-links (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 cross-link 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., Competing Interests: Conflict of interest statement. The authors declare no conflict of interest., (© The Author(s) 2023. Published by Oxford University Press on behalf of American Society of Plant Biologists.)

The proper development of male and female gametophytes is critical for successful sexual reproduction and requires a carefully regulated series of events orchestrated by a suite of various proteins. RUVBL1 and RUVBL2, plant orthologues of human Pontin and Reptin, respectively, belong to the evolutionarily highly conserved AAA + family linked to a wide range of cellular processes. Previously, we found that RUVBL1 and RUVBL2A mutations are homozygous lethal in Arabidopsis. Here, we report that RUVBL1 and RUVBL2A play roles in reproductive development. We show that mutant plants produce embryo sacs with an abnormal structure or with various numbers of nuclei. Although pollen grains of heterozygous mutant plants exhibit reduced viability and reduced pollen tube growth in vitro, some of the ruvbl pollen tubes are capable of targeting ovules in vivo. Similarly, some ruvbl ovules retain the ability to attract wild-type pollen tubes but fail to develop further. The activity of the RUVBL1 and RUVBL2A promoters was observed in the embryo sac, pollen grains, and tapetum cells and, for RUVBL2A, also in developing ovules. In summary, we show that the RUVBL proteins are essential for the proper development of both male and particularly female gametophytes in Arabidopsis., (© 2023 The Authors. The Plant Journal published by Society for Experimental Biology and John Wiley & Sons Ltd.)

The integrity of plant genetic information is constantly challenged by various internal and external factors. Therefore, plants use a sophisticated molecular network to identify, signal and repair damaged DNA. Here, we report on the identification and analysis of four uncharacterized Arabidopsis BRCT5 DOMAIN CONTAINING PROTEINs (BCPs). Proteins with the BRCT5 domain are frequently involved in the maintenance of genome stability across eukaryotes. The screening for sensitivity to induced DNA damage identified BCP1 as the most interesting candidate. We show that BCP1 loss of function mutants are hypersensitive to various types of DNA damage and accumulate an increased number of dead cells in root apical meristems upon DNA damage. Analysis of publicly available sog1 transcriptomic and SOG1 genome-wide DNA binding data revealed that BCP1 is inducible by gamma radiation and is a direct target of this key DNA damage signaling transcription factor. Importantly, bcp1 plants showed a reduced frequency of somatic homologous recombination in response to both endogenous and induced DNA damage. Altogether, we identified a novel plant-specific DNA repair factor that acts downstream of SOG1 in homology-based repair., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Vladejić, Yang, Dvořák Tomaštíková, Doležel, Palecek and Pecinka.)

The Polycomb Repressive Complex 2 (PRC2) is well-known for its role in controlling developmental transitions by suppressing the premature expression of key developmental regulators. Previous work revealed that PRC2 also controls the onset of senescence, a form of developmental programmed cell death (PCD) in plants. Whether the induction of PCD in response to stress is similarly suppressed by the PRC2 remained largely unknown. In this study, we explored whether PCD triggered in response to immunity- and disease-promoting pathogen effectors is associated with changes in the distribution of the PRC2-mediated histone H3 lysine 27 trimethylation (H3K27me3) modification in Arabidopsis thaliana. We furthermore tested the distribution of the heterochromatic histone mark H3K9me2, which is established, to a large extent, by the H3K9 methyltransferase KRYPTONITE, and occupies chromatin regions generally not targeted by PRC2. We report that effector-induced PCD caused major changes in the distribution of both repressive epigenetic modifications and that both modifications have a regulatory role and impact on the onset of PCD during pathogen infection. Our work highlights that the transition to pathogen-induced PCD is epigenetically controlled, revealing striking similarities to developmental PCD., (© The Author(s) 2021. Published by Oxford University Press on behalf of American Society of Plant Biologists.)

Repetitive DNA sequences and some genes are epigenetically repressed by transcriptional gene silencing (TGS). When genetic mutants are not available or problematic to use, TGS can be suppressed by chemical inhibitors. However, informed use of epigenetic inhibitors is partially hampered by the absence of any systematic comparison. In addition, there is emerging evidence that epigenetic inhibitors cause genomic instability, but the nature of this damage and its repair remain unclear. To bridge these gaps, we compared the effects of 5-azacytidine (AC), 2'-deoxy-5-azacytidine (DAC), zebularine and 3-deazaneplanocin A (DZNep) on TGS and DNA damage repair. The most effective inhibitor of TGS was DAC, followed by DZNep, zebularine and AC. We confirmed that all inhibitors induce DNA damage and suggest that this damage is repaired by multiple pathways with a critical role of homologous recombination and of the SMC5/6 complex. A strong positive link between the degree of cytidine analog-induced DNA demethylation and the amount of DNA damage suggests that DNA damage is an integral part of cytidine analog-induced DNA demethylation. This helps us to understand the function of DNA methylation in plants and opens the possibility of using epigenetic inhibitors in biotechnology., (© 2019 The Authors. The Plant Journal © 2019 John Wiley & Sons Ltd.)

TPX2 (Targeting Protein for Xklp2) is an evolutionary conserved microtubule-associated protein important for microtubule nucleation and mitotic spindle assembly. The protein was described as an activator of the mitotic kinase Aurora A in humans and the Arabidopsis AURORA1 (AUR1) kinase. In contrast to animal genomes that encode only one TPX2 gene, higher plant genomes encode a family with several TPX2-LIKE gene members (TPXL). TPXL genes of Arabidopsis can be divided into two groups. Group A proteins (TPXL2, 3, 4, and 8) contain Aurora binding and TPX2_importin domains, while group B proteins (TPXL1, 5, 6, and 7) harbor an Xklp2 domain. Canonical TPX2 contains all the above-mentioned domains. We confirmed using in vitro kinase assays that the group A proteins contain a functional Aurora kinase binding domain. Transient expression of Arabidopsis TPX2-like proteins in Nicotiana benthamiana revealed preferential localization to microtubules and nuclei. Co-expression of AUR1 together with TPX2-like proteins changed the localization of AUR1, indicating that these proteins serve as targeting factors for Aurora kinases. Taken together, we visualize the various localizations of the TPX2-LIKE family in Arabidopsis as a proxy to their functional divergence and provide evidence of their role in the targeted regulation of AUR1 kinase activity.

Several histone variants are posttranslationally phosphorylated. Little is known about phosphorylation of the centromere-specific histone 3 (CENH3) variant in plants. We show that CENH3 of Arabidopsis thaliana is phosphorylated in vitro by Aurora3, predominantly at serine 65. Interaction of Aurora3 and CENH3 was found by immunoprecipitation (IP) in A. thaliana and by bimolecular fluorescence complementation. Western blotting with an anti-CENH3 pS65 antibody showed that CENH3 pS65 is more abundant in flower buds than elsewhere in the plant. Substitution of serine 65 by either alanine or aspartic acid resulted in a range of phenotypic abnormalities, especially in reproductive tissues. We conclude that Aurora3 phosphorylates CENH3 at S65 and that this post-translational modification is required for the proper development of the floral meristem.