An E3 Ubiquitin Ligase Localization Screen Uncovers DTX2 as a Novel ADP-Ribosylation-Dependent Regulator of DNA Double-Strand Break Repair.

DNA double-strand breaks (DSBs) elicit an elaborate response to signal damage and trigger repair via two major pathways: non-homologous end-joining (NHEJ), which functions throughout the interphase, and homologous recombination (HR), restricted to S/G2 phases. The DNA damage response (DDR) relies, on post-translational modifications of nuclear factors to coordinate the mending of breaks. Ubiquitylation of histones and chromatin-associated factors regulates DSB repair and numerous E3 ubiquitin ligases are involved in this process. Despite significant progress, our understanding of ubiquitin-mediated DDR regulation remains incomplete. Here, we have performed a localization screen to identify RING/U-box E3 ligases involved in genome maintenance. Our approach uncovered 7 novel E3 ligases that are recruited to microirradiation stripes, suggesting potential roles in DNA damage signaling and repair. Amongst these factors, the DELTEX family E3 ligase DTX2 is rapidly mobilized to lesions in a poly ADP-ribosylation-dependent manner. DTX2 is recruited and retained at DSBs via its WWE and DTC domains. In cells, both domains are required for optimal binding to mono and poly ADP-ribosylated proteins with WWEs playing a prominent role in this process. Supporting its involvement in DSB repair, DTX2 depletion decreases HR efficiency and moderately enhances NHEJ. Furthermore, DTX2 depletion impeded BRCA1 foci formation and increased 53BP1 accumulation at DSBs, suggesting a fine-tuning role for this E3 ligase in repair pathway choice. Finally, DTX2 depletion sensitized cancer cells to X-rays and PARP inhibition and these susceptibilities could be rescued by DTX2 re-expression. Altogether, our work identifies DTX2 as a novel ADP-ribosylation-dependent regulator of HR-mediated DSB repair.

SMARCAL1 ubiquitylation controls its association with RPA-coated ssDNA and promotes replication fork stability.

Impediments in replication fork progression cause genomic instability, mutagenesis, and severe pathologies. At stalled forks, RPA-coated single-stranded DNA (ssDNA) activates the ATR kinase and directs fork remodeling, 2 key early events of the replication stress response. RFWD3, a recently described Fanconi anemia (FA) ubiquitin ligase, associates with RPA and promotes its ubiquitylation, facilitating late steps of homologous recombination (HR). Intriguingly, RFWD3 also regulates fork progression, restart and stability via poorly understood mechanisms. Here, we used proteomics to identify putative RFWD3 substrates during replication stress in human cells. We show that RFWD3 interacts with and ubiquitylates the SMARCAL1 DNA translocase directly in vitro and following DNA damage in vivo. SMARCAL1 ubiquitylation does not trigger its subsequent proteasomal degradation but instead disengages it from RPA thereby regulating its function at replication forks. Proper regulation of SMARCAL1 by RFWD3 at stalled forks protects them from excessive MUS81-mediated cleavage in response to UV irradiation, thereby limiting DNA replication stress. Collectively, our results identify RFWD3-mediated SMARCAL1 ubiquitylation as a novel mechanism that modulates fork remodeling to avoid genome instability triggered by aberrant fork processing.

Pseudophosphorylation of Arabidopsis jasmonate biosynthesis enzyme lipoxygenase 2 via mutation of Ser600 inhibits enzyme activity.

Jasmonates are oxylipin phytohormones critical for plant resistance against necrotrophic pathogens and chewing herbivores. An early step in their biosynthesis is catalyzed by non-heme iron lipoxygenases (LOX; EC 1.13.11.12). In Arabidopsis thaliana, phosphorylation of Ser600 of AtLOX2 was previously reported, but whether phosphorylation regulates AtLOX2 activity is unclear. Here, we characterize the kinetic properties of recombinant WT AtLOX2 (AtLOX2WT). AtLOX2WT displays positive cooperativity with α-linolenic acid (α-LeA, jasmonate precursor), linoleic acid (LA), and arachidonic acid (AA) as substrates. Enzyme velocity with endogenous substrates α-LeA and LA increased with pH. For α-LeA, this increase was accompanied by a decrease in substrate affinity at alkaline pH; thus, the catalytic efficiency for α-LeA was not affected over the pH range tested. Analysis of Ser600 phosphovariants demonstrated that pseudophosphorylation inhibits enzyme activity. AtLOX2 activity was not detected in phosphomimics Atlox2S600D and Atlox2S600M when α-LeA or AA were used as substrates. In contrast, phosphonull mutant Atlox2S600A exhibited strong activity with all three substrates, α-LeA, LA, and AA. Structural comparison between the AtLOX2 AlphaFold model and a complex between 8R-LOX and a 20C polyunsaturated fatty acid suggests a close proximity between AtLOX2 Ser600 and the carboxylic acid head group of the polyunsaturated fatty acid. This analysis indicates that Ser600 is located at a critical position within the AtLOX2 structure and highlights how Ser600 phosphorylation could affect AtLOX2 catalytic activity. Overall, we propose that AtLOX2 Ser600 phosphorylation represents a key mechanism for the regulation of AtLOX2 activity and, thus, the jasmonate biosynthesis pathway and plant resistance.

Zinc controls PML nuclear body formation through regulation of a paralog specific auto-inhibition in SUMO1.

SUMO proteins are important regulators of many key cellular functions in part through their ability to form interactions with other proteins containing SUMO interacting motifs (SIMs). One characteristic feature of all SUMO proteins is the presence of a highly divergent intrinsically disordered region at their N-terminus. In this study, we examine the role of this N-terminal region of SUMO proteins in SUMO-SIM interactions required for the formation of nuclear bodies by the promyelocytic leukemia (PML) protein (PML-NBs). We demonstrate that the N-terminal region of SUMO1 functions in a paralog specific manner as an auto-inhibition domain by blocking its binding to the phosphorylated SIMs of PML and Daxx. Interestingly, we find that this auto-inhibition in SUMO1 is relieved by zinc, and structurally show that zinc stabilizes the complex between SUMO1 and a phospho-mimetic form of the SIM of PML. In addition, we demonstrate that increasing cellular zinc levels enhances PML-NB formation in senescent cells. Taken together, these results provide important insights into a paralog specific function of SUMO1, and suggest that zinc levels could play a crucial role in regulating SUMO1-SIM interactions required for PML-NB formation and function.

Membrane Targeting and GTPase Activity of Rab7 Are Required for Its Ubiquitination by RNF167.

Rab7 is a GTPase that controls late endosome and lysosome trafficking. Recent studies have demonstrated that Rab7 is ubiquitinated, a post-translational modification mediated by an enzymatic cascade. To date, only one ubiquitin E3 ligase and one deubiquitinase have been identified in regulating Rab7 ubiquitination. Here, we report that RNF167, a transmembrane endolysosomal ubiquitin ligase, can ubiquitinate Rab7. Using immunoprecipitation and in vitro ubiquitination assays, we demonstrate that Rab7 is a direct substrate of RNF167. Subcellular fractionation indicates that RNF167 activity maintains Rab7's membrane localization. Epifluorescence microscopy in HeLa cells shows that Rab7-positive vesicles are larger under conditions enabling Rab7 ubiquitination by RNF167. Characterization of its ubiquitination reveals that Rab7 must be in its GTP-bound active form for membrane anchoring and, thus, accessible for RNF167-mediated ubiquitin attachment. Cellular distribution analyses of lysosome marker Lamp1 show that vesicle positioning is independent of Rab7 and RNF167 expression and that Rab7 endosomal localization is not affected by RNF167 knockdown. However, both Rab7 and RNF167 depletion affect each other's lysosomal localization. Finally, this study demonstrates that the RNF167-mediated ubiquitination of Rab7 GTPase is impaired by variants of Charcot-Marie-Tooth Type 2B disease. This study identified RNF167 as a new ubiquitin ligase for Rab7 while expanding our knowledge of the mechanisms underlying the ubiquitination of Rab7.

RNF13 Dileucine Motif Variants L311S and L312P Interfere with Endosomal Localization and AP-3 Complex Association.

Developmental and epileptic encephalopathies (DEE) are rare and serious neurological disorders characterized by severe epilepsy with refractory seizures and a significant developmental delay. Recently, DEE73 was linked to genetic alterations of the RNF13 gene, which convert positions 311 or 312 in the RNF13 protein from leucine to serine or proline, respectively (L311S and L312P). Using a fluorescence microscopy approach to investigate the molecular and cellular mechanisms affected by RNF13 protein variants, the current study shows that wild-type RNF13 localizes extensively with endosomes and lysosomes, while L311S and L312P do not extensively colocalize with the lysosomal marker Lamp1. Our results show that RNF13 L311S and L312P proteins affect the size of endosomal vesicles along with the temporal and spatial progression of fluorescently labeled epidermal growth factor, but not transferrin, in the endolysosomal system. Furthermore, GST-pulldown and co-immunoprecipitation show that RNF13 variants disrupt association with AP-3 complex. Knockdown of AP-3 complex subunit AP3D1 alters the lysosomal localization of wild-type RNF13 and similarly affects the size of endosomal vesicles. Importantly, our study provides a first step toward understanding the cellular and molecular mechanism altered by DEE73-associated genetic variations of RNF13.

DNA asymmetry promotes SUMO modification of the single-stranded DNA-binding protein RPA.

Repair of DNA double-stranded breaks by homologous recombination (HR) is dependent on DNA end resection and on post-translational modification of repair factors. In budding yeast, single-stranded DNA is coated by replication protein A (RPA) following DNA end resection, and DNA-RPA complexes are then SUMO-modified by the E3 ligase Siz2 to promote repair. Here, we show using enzymatic assays that DNA duplexes containing 3' single-stranded DNA overhangs increase the rate of RPA SUMO modification by Siz2. The SAP domain of Siz2 binds DNA duplexes and makes a key contribution to this process as highlighted by models and a crystal structure of Siz2 and by assays performed using protein mutants. Enzymatic assays performed using DNA that can accommodate multiple RPA proteins suggest a model in which the SUMO-RPA signal is amplified by successive rounds of Siz2-dependent SUMO modification of RPA and dissociation of SUMO-RPA at the junction between single- and double-stranded DNA. Our results provide insights on how DNA architecture scaffolds a substrate and E3 ligase to promote SUMO modification in the context of DNA repair.

Plant SUMO E3 Ligases: Function, Structural Organization, and Connection With DNA.

Protein modification by the small ubiquitin-like modifier (SUMO) plays an important role in multiple plant processes, including growth, development, and the response to abiotic stresses. Mechanistically, SUMOylation is a sequential multi-enzymatic process where SUMO E3 ligases accelerate SUMO conjugation while also influencing target identity and interactions. This review explores the biological functions of plant SUMO E3 ligases [SAP AND MIZ1 DOMAIN-CONTAINING LIGASE (SIZs), METHYL METHANESULFONATE-SENSITIVITY PROTEIN 21 (MMS21s), and PROTEIN INHIBITOR OF ACTIVATED STAT-LIKE (PIALs)] in relation to their molecular activities and domains. We also explore the sub-cellular localization of SUMO E3 ligases and review evidence suggesting a connection between certain SUMO E3 ligases and DNA that contributes to gene expression regulation.

Functional interaction of ubiquitin ligase RNF167 with UBE2D1 and UBE2N promotes ubiquitination of AMPA receptor.

Protein ubiquitination has been historically associated with protein degradation, but recent studies have demonstrated other cellular functions associated with substrate ubiquitination. Among the RING-type ubiquitin E3 ligase enzymes present in the human genome, RNF167 is a transmembrane protein located in endosomes and lysosomes and is implicated in controlling the endolysosomal pathway. Substrates of RNF167 have been identified, but the ubiquitin-conjugating E2 enzymes involved in the mechanism remain unknown. In this study, we describe the interaction between RNF167 and conjugating E2 enzymes. By means of in vitro autoubiquitination and binding assays, we show that RNF167 functionally interacts with many conjugating E2s, while fluorescence microscopy illustrates that these interactions occur in endosomes and lysosomes. Kinetic analyses of the interaction between RNF167 and selected conjugating E2 enzymes reveal submicromolar dissociation constants. The computed model of interaction between the RING domain of RNF167 and conjugating enzymes gives us insights on how RNF167 could interact with conjugating E2 enzymes. Furthermore, the results reveal that in vitro polyubiquitination of the AMPA-type glutamate receptor subunit GluA2, one of the RNF167's known substrates, is possible by the conjugating E2 enzyme UBE2N only after GluA2 has been primed by ubiquitin subsequent to the action of an initiating conjugating E2 enzyme functionally binding RNF167. Pharmacological inhibition of UBE2N in cultured hippocampal neurons diminishes AMPA-induced GluA2 ubiquitination. This study characterizes interacting partners of RNF167 and constitutes an initial step toward the identification of functional pairs assembled from RNF167 and ubiquitin-conjugating E2 enzymes involved in the ubiquitination of RNF167's substrate.

Functional targeting of the TGF-ßR1 kinase domain and downstream signaling: A role for the galloyl moiety of green tea-derived catechins in ES-2 ovarian clear cell carcinoma.

The galloyl moiety is a specific structural feature which dictates, in part, the chemopreventive properties of diet-derived catechins. In ovarian cancer cells, galloylated catechins were recently demonstrated to target the transforming growth factor (TGF)-ß-mediated control of the epithelial-mesenchymal transition process. The specific impact of the galloyl moiety on such signaling, however, remains poorly understood. Here, we questioned whether the sole galloyl moiety interacted with TGF-ß-receptors to alter signal transduction and chemotactic migratory response in an ES-2 serous carcinoma-derived ovarian cancer cell model. In line with the LogP and LogS values of the tested molecules, we found that TGF-ß-induced Smad-3 phosphorylation and cell migration were optimally inhibited, provided that the lateral aliphatic chain of the galloyl moiety reached 8-10 carbons. Functional inhibition of the TGF-ß receptor (TGF-ßR1) kinase activity was supported by surface plasmon resonance assays showing direct physical interaction between TGF-ßR1 and the galloyl moiety. In silico molecular docking analysis predicted a model where galloylated catechins may bind TGF-ßR1 within its adenosine triphosphate binding cleft in a site analogous to that of Galunisertib, a selective adenosine triphosphate-mimetic competitive inhibitor of TGF-ßR1. In conclusion, our data suggest that the galloyl moiety of the diet-derived catechins provides specificity of action to galloylated catechins by positioning them within the kinase domain of the TGF-ßR1 in order to antagonize TGF-ß-mediated signaling that is required for ovarian cancer cell invasion and metastasis.

Characterization of a C-Terminal SUMO-Interacting Motif Present in Select PIAS-Family Proteins.

The human PIAS proteins are small ubiquitin-like modifier (SUMO) E3 ligases that participate in important cellular functions. Several of these functions depend on a conserved SUMO-interacting motif (SIM) located in the central region of all PIAS proteins (SIM1). Recently, it was determined that Siz2, a yeast homolog of PIAS proteins, possesses a second SIM at its C terminus (SIM2). Sequence alignment indicates that a SIM2 is also present in PIAS1-3, but not PIAS4. Using biochemical and structural studies, we demonstrate PIAS-SIM2 binds to SUMO1, but that phosphorylation of the PIAS-SIM2 or acetylation of SUMO1 alter this interaction in a manner distinct from what is observed for the PIAS-SIM1. We also show that the PIAS-SIM2 plays a key role in formation of a UBC9-PIAS1-SUMO1 complex. These results provide insights into how post-translational modifications selectively regulate the specificity of multiple SIMs found in the PIAS proteins by exploiting the plasticity built into the SUMO-SIM binding interface.

Acetylation of SUMO1 Alters Interactions with the SIMs of PML and Daxx in a Protein-Specific Manner.

The interactions between SUMO proteins and SUMO-interacting motif (SIM) in nuclear bodies formed by the promyelocytic leukemia (PML) protein (PML-NBs) have been shown to be modulated by either phosphorylation of the SIMs or acetylation of SUMO proteins. However, little is known about how this occurs at the atomic level. In this work, we examined the role that acetylation of SUMO1 plays on its binding to the phosphorylated SIMs (phosphoSIMs) of PML and Daxx. Our results demonstrate that SUMO1 binding to the phosphoSIM of either PML or Daxx is dramatically reduced by acetylation at either K39 or K46. However, acetylation at K37 only impacts binding to Daxx. Structures of acetylated SUMO1 variants bound to the phosphoSIMs of PML and Daxx demonstrate that there is structural plasticity in SUMO-SIM interactions. The plasticity observed in these structures provides a robust mechanism for regulating SUMO-SIM interactions in PML-NBs using signaling generated post-translational modifications.

Ubiquitin-like Protein Conjugation: Structures, Chemistry, and Mechanism.

Ubiquitin-like proteins (Ubl's) are conjugated to target proteins or lipids to regulate their activity, stability, subcellular localization, or macromolecular interactions. Similar to ubiquitin, conjugation is achieved through a cascade of activities that are catalyzed by E1 activating enzymes, E2 conjugating enzymes, and E3 ligases. In this review, we will summarize structural and mechanistic details of enzymes and protein cofactors that participate in Ubl conjugation cascades. Precisely, we will focus on conjugation machinery in the SUMO, NEDD8, ATG8, ATG12, URM1, UFM1, FAT10, and ISG15 pathways while referring to the ubiquitin pathway to highlight common or contrasting themes. We will also review various strategies used to trap intermediates during Ubl activation and conjugation.

A phosphorylation-and-ubiquitylation circuitry driving ATR activation and homologous recombination.

RPA-coated single-stranded DNA (RPA-ssDNA), a nucleoprotein structure induced by DNA damage, promotes ATR activation and homologous recombination (HR). RPA is hyper-phosphorylated and ubiquitylated after DNA damage. The ubiquitylation of RPA by PRP19 and RFWD3 facilitates ATR activation and HR, but how it is stimulated by DNA damage is still unclear. Here, we show that RFWD3 binds RPA constitutively, whereas PRP19 recognizes RPA after DNA damage. The recruitment of PRP19 by RPA depends on PIKK-mediated RPA phosphorylation and a positively charged pocket in PRP19. An RPA32 mutant lacking phosphorylation sites fails to recruit PRP19 and support RPA ubiquitylation. PRP19 mutants unable to bind RPA or lacking ubiquitin ligase activity also fail to support RPA ubiquitylation and HR. These results suggest that RPA phosphorylation enhances the recruitment of PRP19 to RPA-ssDNA and stimulates RPA ubiquitylation through a process requiring both PRP19 and RFWD3, thereby triggering a phosphorylation-ubiquitylation circuitry that promotes ATR activation and HR.

Molecular basis of interactions between SH3 domain-containing proteins and the proline-rich region of the ubiquitin ligase Itch.

The ligase Itch plays major roles in signaling pathways by inducing ubiquitylation-dependent degradation of several substrates. Substrate recognition and binding are critical for the regulation of this reaction. Like closely related ligases, Itch can interact with proteins containing a PPXY motif via its WW domains. In addition to these WW domains, Itch possesses a proline-rich region (PRR) that has been shown to interact with several Src homology 3 (SH3) domain-containing proteins. We have previously established that despite the apparent surface uniformity and conserved fold of SH3 domains, they display different binding mechanisms and affinities for their interaction with the PRR of Itch. Here, we attempt to determine the molecular bases underlying the wide range of binding properties of the Itch PRR. Using pulldown assays combined with mass spectrometry analysis, we show that the Itch PRR preferentially forms complexes with endophilins, amphyphisins, and pacsins but can also target a variety of other SH3 domain-containing proteins. In addition, we map the binding sites of these proteins using a combination of PRR sub-sequences and mutants. We find that different SH3 domains target distinct proline-rich sequences overlapping significantly. We also structurally analyze these protein complexes using crystallography and molecular modeling. These structures depict the position of Itch PRR engaged in a 1:2 protein complex with ß-PIX and a 1:1 complex with the other SH3 domain-containing proteins. Taken together, these results reveal the binding preferences of the Itch PRR toward its most common SH3 domain-containing partners and demonstrate that the PRR region is sufficient for binding.

Structural and Biochemical Characterization of a Copper-Binding Mutant of the Organomercurial Lyase MerB: Insight into the Key Role of the Active Site Aspartic Acid in Hg-Carbon Bond Cleavage and Metal Binding Specificity.

In bacterial resistance to mercury, the organomercurial lyase (MerB) plays a key role in the detoxification pathway through its ability to cleave Hg-carbon bonds. Two cysteines (C96 and C159; Escherichia coli MerB numbering) and an aspartic acid (D99) have been identified as the key catalytic residues, and these three residues are conserved in all but four known MerB variants, where the aspartic acid is replaced with a serine. To understand the role of the active site serine, we characterized the structure and metal binding properties of an E. coli MerB mutant with a serine substituted for D99 (MerB D99S) as well as one of the native MerB variants containing a serine residue in the active site (Bacillus megaterium MerB2). Surprisingly, the MerB D99S protein copurified with a bound metal that was determined to be Cu(II) from UV-vis absorption, inductively coupled plasma mass spectrometry, nuclear magnetic resonance, and electron paramagnetic resonance studies. X-ray structural studies revealed that the Cu(II) is bound to the active site cysteine residues of MerB D99S, but that it is displaced following the addition of either an organomercurial substrate or an ionic mercury product. In contrast, the B. megaterium MerB2 protein does not copurify with copper, but the structure of the B. megaterium MerB2-Hg complex is highly similar to the structure of the MerB D99S-Hg complexes. These results demonstrate that the active site aspartic acid is crucial for both the enzymatic activity and metal binding specificity of MerB proteins and suggest a possible functional relationship between MerB and its only known structural homologue, the copper-binding protein NosL.

Soluble expression, purification and functional characterization of a coil peptide composed of a positively charged and hydrophobic motif.

A de novo heterodimeric coiled-coil system formed by the association of two synthetic peptides, the Ecoil and Kcoil, has been previously designed and proven to be an excellent and versatile tool for various biotechnology applications. However, based on the challenges encountered during its chemical synthesis, the Kcoil peptide has been designated as a "difficult peptide". In this study, we explore the expression of the Kcoil peptide by a bacterial system as well as its subsequent purification. The maximum expression level was observed when the peptide was fused to thioredoxin and the optimized purification process consisted of three chromatographic steps: immobilized-metal affinity chromatography followed by cation-exchange chromatography and, finally, a reverse-phase high-performance liquid chromatography. This entire process led to a final volumetric production yield of 1.5 mg of pure Kcoil peptide per liter of bacterial culture, which represents a significant step towards the cost-effective production and application of coiled-coil motifs. Our results thus demonstrate for the first time that bacterial production is a viable alternative to the chemical synthesis of de novo designed coil peptides.

Structural basis for catalytic activation by the human ZNF451 SUMO E3 ligase.

E3 protein ligases enhance transfer of ubiquitin-like (Ubl) proteins from E2 conjugating enzymes to substrates by stabilizing the thioester-charged E2~Ubl in a closed configuration optimally aligned for nucleophilic attack. Here, we report biochemical and structural data that define the N-terminal domain of the Homo sapiens ZNF451 as the catalytic module for SUMO E3 ligase activity. The ZNF451 catalytic module contains tandem SUMO-interaction motifs (SIMs) bridged by a Pro-Leu-Arg-Pro (PLRP) motif. The first SIM and PLRP motif engage thioester-charged E2~SUMO while the next SIM binds a second molecule of SUMO bound to the back side of E2. We show that ZNF451 is SUMO2 specific and that SUMO modification of ZNF451 may contribute to activity by providing a second molecule of SUMO that interacts with E2. Our results are consistent with ZNF451 functioning as a bona fide SUMO E3 ligase.

Structural and functional characterization of the phosphorylation-dependent interaction between PML and SUMO1.

PML and several other proteins localizing in PML-nuclear bodies (PML-NB) contain phosphoSIMs (SUMO-interacting motifs), and phosphorylation of this motif plays a key role in their interaction with SUMO family proteins. We examined the role that phosphorylation plays in the binding of the phosphoSIMs of PML and Daxx to SUMO1 at the atomic level. The crystal structures of SUMO1 bound to unphosphorylated and tetraphosphorylated PML-SIM peptides indicate that three phosphoserines directly contact specific positively charged residues of SUMO1. Surprisingly, the crystal structure of SUMO1 bound to a diphosphorylated Daxx-SIM peptide indicate that the hydrophobic residues of the phosphoSIM bind in a manner similar to that seen with PML, but important differences are observed when comparing the phosphorylated residues. Together, the results provide an atomic level description of how specific acetylation patterns within different SUMO family proteins can work together with phosphorylation of phosphoSIM's regions of target proteins to regulate binding specificity.

A family portrait: structural comparison of the Whirly proteins from Arabidopsis thaliana and Solanum tuberosum.

DNA double-strand breaks are highly detrimental genomic lesions that routinely arise in genomes. To protect the integrity of their genetic information, all organisms have evolved specialized DNA-repair mechanisms. Whirly proteins modulate DNA repair in plant chloroplasts and mitochondria by binding single-stranded DNA in a non-sequence-specific manner. Although most of the results showing the involvement of the Whirly proteins in DNA repair have been obtained in Arabidopsis thaliana, only the crystal structures of the potato Whirly proteins WHY1 and WHY2 have been reported to date. The present report of the crystal structures of the three Whirly proteins from A. thaliana (WHY1, WHY2 and WHY3) reveals that these structurally similar proteins assemble into tetramers. Furthermore, structural alignment with a potato WHY2-DNA complex reveals that the residues in these proteins are properly oriented to bind single-stranded DNA in a non-sequence-specific manner.