SAP Domain Research Articles

Role of the SAF-A SAP domain in X inactivation, transcription, splicing, and cell proliferation.

SAF-A is conserved throughout vertebrates and has emerged as an important factor regulating a multitude of nuclear functions, including lncRNA localization, gene expression, and splicing. SAF-A has several functional domains, including an N-terminal SAP domain that binds directly to DNA. Phosphorylation of SAP domain serines S14 and S26 are important for SAF-A localization and function during mitosis, however whether these serines are involved in interphase functions of SAF-A is not known. In this study we tested for the role of the SAP domain, and SAP domain serines S14 and S26 in X chromosome inactivation, protein dynamics, gene expression, splicing, and cell proliferation. Here we show that the SAP domain serines S14 and S26 are required to maintain XIST RNA localization and polycomb-dependent histone modifications on the inactive X chromosome in female cells. In addition, we present evidence that an Xi localization signal resides in the SAP domain. We found that that the SAP domain is not required to maintain gene expression and plays only a minor role in mRNA splicing. In contrast, the SAF-A SAP domain, in particular serines S14 and S26, are required for normal protein dynamics, and to maintain normal cell proliferation. We propose a model whereby dynamic phosphorylation of SAF-A serines S14 and S26 mediates rapid turnover of SAF-A interactions with DNA during interphase.

Exploring the structural landscape of DNA maintenance proteins

Evolutionary annotation of genome maintenance (GM) proteins has conventionally been established by remote relationships within protein sequence databases. However, often no significant relationship can be established. Highly sensitive approaches to attain remote homologies based on iterative profile-to-profile methods have been developed. Still, these methods have not been systematically applied in the evolutionary annotation of GM proteins. Here, by applying profile-to-profile models, we systematically survey the repertoire of GM proteins from bacteria to man. We identify multiple GM protein candidates and annotate domains in numerous established GM proteins, among other PARP, OB-fold, Macro, TUDOR, SAP, BRCT, KU, MYB (SANT), and nuclease domains. We experimentally validate OB-fold and MIS18 (Yippee) domains in SPIDR and FAM72 protein families, respectively. Our results indicate that, surprisingly, despite the immense interest and long-term research efforts, the repertoire of genome stability caretakers is still not fully appreciated.

The KU70-SAP domain has an overlapping function with DNA-PKcs in limiting the lateral movement of KU along DNA.

Ku70 is a conserved non-homologous end-joining (NHEJ) factor. Using genetically engineered mouse models and biochemical analyses, our study uncovered a previously unappreciated role of the C-terminal SAP domain of Ku70 in limiting the lateral movement of KU on DNA ends and ensuring end protection. The presence of DNA-PKcs partially masks this role of the SAP domain.

Structural basis for RAD18 regulation by MAGEA4 and its implications for RING ubiquitin ligase binding by MAGE family proteins

MAGEA4 is a cancer-testis antigen primarily expressed in the testes but aberrantly overexpressed in several cancers. MAGEA4 interacts with the RING ubiquitin ligase RAD18 and activates trans-lesion DNA synthesis (TLS), potentially favouring tumour evolution. Here, we employed NMR and AlphaFold2 (AF) to elucidate the interaction mode between RAD18 and MAGEA4, and reveal that the RAD6-binding domain (R6BD) of RAD18 occupies a groove in the C-terminal winged-helix subdomain of MAGEA4. We found that MAGEA4 partially displaces RAD6 from the RAD18 R6BD and inhibits degradative RAD18 autoubiquitination, which could be countered by a competing peptide of the RAD18 R6BD. AlphaFold2 and cross-linking mass spectrometry (XL-MS) also revealed an evolutionary invariant intramolecular interaction between the catalytic RING and the DNA-binding SAP domains of RAD18, which is essential for PCNA mono-ubiquitination. Using interaction proteomics, we found that another Type-I MAGE, MAGE-C2, interacts with the RING ubiquitin ligase TRIM28 in a manner similar to the MAGEA4/RAD18 complex, suggesting that the MAGEA4 peptide-binding groove also serves as a ligase-binding cleft in other type-I MAGEs. Our data provide new insights into the mechanism and regulation of RAD18-mediated PCNA mono-ubiquitination.

The SAP domain of Ku facilitates its efficient loading onto DNA ends.

The evolutionarily conserved DNA repair complex Ku serves as the primary sensor of free DNA ends in eukaryotic cells. Its rapid association with DNA ends is crucial for several cellular processes, including non-homologous end joining (NHEJ) DNA repair and telomere protection. In this study, we conducted a transient kinetic analysis to investigate the impact of the SAP domain on individual phases of the Ku-DNA interaction. Specifically, we examined the initial binding, the subsequent docking of Ku onto DNA, and sliding of Ku along DNA. Our findings revealed that the C-terminal SAP domain of Ku70 facilitates the initial phases of the Ku-DNA interaction but does not affect the sliding process. This suggests that the SAP domain may either establish the first interactions with DNA, or stabilize these initial interactions during loading. To assess the biological role of the SAP domain, we generated Arabidopsis plants expressing Ku lacking the SAP domain. Intriguingly, despite the decreased efficiency of the ΔSAP Ku complex in loading onto DNA, the mutant plants exhibited full proficiency in classical NHEJ and telomere maintenance. This indicates that the speed with which Ku loads onto telomeres or DNA double-strand breaks is not the decisive factor in stabilizing these DNA structures.

Selective DNA-binding of SP120 (rat ortholog of human hnRNP U) is mediated by arginine-glycine rich domain and modulated by RNA.

A human protein heterogeneous ribonucleoprotein U (hnRNP U) also known as Scaffold attachment factor A (SAF-A) and its orthologous rat protein SP120 are abundant and multifunctional nuclear protein that directly binds to both DNA and RNA. The C-terminal region of hnRNP U enriched with arginine and glycine is essential for the interaction with RNA and the N-terminal region of SAF-A termed SAP domain has been ascribed to the DNA binding. We have reported that rat hnRNP U specifically and cooperatively binds to AT-rich DNA called nuclear scaffold/matrix-associated region (S/MAR) although its detailed mechanism remained unclear. In the present study analysis of hnRNP U deletion mutants revealed for the first time that a C-terminal domain enriched with Arg-Gly (defined here as 'RG domain') is predominantly important for the S/MAR-selective DNA binding activities. RG domain alone directly bound to S/MAR and coexistence with the SAP domain exerted a synergistic effect. The binding was inhibited by netropsin, a minor groove binder with preference to AT pairs that are enriched in S/MAR, suggesting that RG domain interacts with minor groove of S/MAR DNA. Interestingly, excess amounts of RNA attenuated the RG domain-dependent S/MAR-binding of hnRNP U. Taken together, hnRNP U may be the key element for the RNA-regulated recognition of S/MAR DNA and thus contributing to the dynamic structural changes of chromatin compartments.

Insight into the Structural Basis for Dual Nucleic Acid-Recognition by the Scaffold Attachment Factor B2 Protein.

The family of scaffold attachment factor B (SAFB) proteins comprises three members and was first identified as binders of the nuclear matrix/scaffold. Over the past two decades, SAFBs were shown to act in DNA repair, mRNA/(l)ncRNA processing and as part of protein complexes with chromatin-modifying enzymes. SAFB proteins are approximately 100 kDa-sized dual nucleic acid-binding proteins with dedicated domains in an otherwise largely unstructured context, but whether and how they discriminate DNA and RNA binding has remained enigmatic. We here provide the SAFB2 DNA- and RNA-binding SAP and RRM domains in their functional boundaries and use solution NMR spectroscopy to ascribe DNA- and RNA-binding functions. We give insight into their target nucleic acid preferences and map the interfaces with respective nucleic acids on sparse data-derived SAP and RRM domain structures. Further, we provide evidence that the SAP domain exhibits intra-domain dynamics and a potential tendency to dimerize, which may expand its specifically targeted DNA sequence range. Our data provide a first molecular basis of and a starting point towards deciphering DNA- and RNA-binding functions of SAFB2 on the molecular level and serve a basis for understanding its localization to specific regions of chromatin and its involvement in the processing of specific RNA species.

Apple SUMO E3 ligase MdSIZ1 regulates cuticular wax biosynthesis by SUMOylating transcription factor MdMYB30.

A key function of SUMOylation is the coordinated modification of numerous proteins to optimize plant growth and resistance to environmental stress. Plant cuticular wax is deposited on the surface of primary plant organs to form a barrier that provides protection against changes in terrestrial environments. Many recent studies have examined cuticular wax biosynthetic pathways and regulation. However, whether SUMOylation is involved in the regulation of cuticle wax deposition at the posttranslational level remains unclear. Here, we demonstrate that a small ubiquitin-like modifier (SUMO) E3 ligase, SAP AND MIZ1 DOMAIN CONTAINING LIGASE1 (MdSIZ1), regulates wax accumulation and cuticle permeability in apple (Malus domestica Borkh), SUMO E2 CONJUGATING ENZYME 1(MdSCE1) physically interacts with MdMYB30, a transcription factor involved in the regulation of cuticle wax accumulation. MdSIZ1 mediates the SUMOylation and accumulation of MdMYB30 by inhibiting its degradation through the 26S proteasome pathway. Furthermore, MdMYB30 directly binds to the β-KETOACYL-COA SYNTHASE 1 (MdKCS1) promoter to activate its expression and promote wax biosynthesis. These findings indicate that the MdSIZ1-MdMYB30-MdKCS1 module positively regulates cuticular wax biosynthesis in apples. Overall, the findings of our study provide insights into the regulation pathways involved in cuticular wax biosynthesis.

SAF-A/hnRNP U binds polyphosphoinositides via a lysine rich polybasic motif located in the SAP domain.

Polyphosphoinositides (PPIn) play essential functions as lipid signalling molecules and many of their functions have been elucidated in the cytoplasm. However, PPIn are also intranuclear where they contribute to chromatin remodelling, transcription and mRNA splicing. Using quantitative interactomics, we have previously identified PPIn-interacting proteins with roles in RNA processing/splicing including the heterogeneous nuclear ribonucleoprotein U (hnRNPU/SAF-A). In this study, hnRNPU was validated as a direct PPIn-interacting protein via 2 regions located in the N and C termini. Furthermore, deletion of the polybasic motif region located at aa 9-24 in its DNA binding SAP domain prevented PPIn interaction. In conclusion, these results are consistent with hnRNPU harbouring a polybasic region with dual functions in DNA and PPIn interaction.

Phosphorylation of the DNA repair scaffold SLX4 drives folding of the SAP domain and activation of the MUS81-EME1 endonuclease

The DNA repair scaffold SLX4 has multifaceted roles in genome stability, many of which depend on structure-selective endonucleases. SLX4 coordinates the cell cycle-regulated assembly of SLX1, MUS81-EME1, and XPF-ERCC1 into a tri-nuclease complex called SMX. Mechanistically, how the mitotic kinase CDK1 regulates the interaction between SLX4 and MUS81-EME1 remains unclear. Here, we show that CDK1-cyclin B phosphorylates SLX4 residues T1544, T1561, and T1571 in the MUS81-binding region (SLX4MBR). Phosphorylated SLX4MBR relaxes the substrate specificity of MUS81-EME1 and stimulates cleavage of replication and recombination structures, providing a biochemical explanation for the chromosome pulverization that occurs when SLX4 binds MUS81 in S-phase. Remarkably, phosphorylation of SLX4MBR drives folding of an SAP domain, which underpins the high-affinity interaction with MUS81. We also report the structure of phosphorylated SLX4MBR and identify the MUS81-binding interface. Our work provides mechanistic insights into how cell cycle-regulated phosphorylation of SLX4 drives the recruitment and activation of MUS81-EME1.

Pepper SUMO E3 ligase CaDSIZ1 enhances drought tolerance by stabilizing the transcription factor CaDRHB1.

Small ubiquitin-like modifier (SUMO) conjugation (SUMOylation) is a reversible post-translational modification associated with protein stability and activity, and modulates hormone signaling and stress responses in plants. Previously, we reported that the pepper dehydration-responsive homeobox domain transcription factor CaDRHB1 acts as a positive modulator of drought response. Here, we show that CaDRHB1 protein stability is enhanced by SUMO E3 ligase Capsicum annuum DRHB1-interacting SAP and Miz domain (SIZ1) (CaDSIZ1)-mediated SUMOylation in response to drought, thereby positively modulating abscisic acid (ABA) signaling and drought responses. Substituting lysine (K) 138 of CaDRHB1 with arginine reduced CaDSIZ1-mediated SUMOylation, indicating that K138 is the principal site for SUMO conjugation. Virus-induced silencing of CaDSIZ1 promoted CaDRHB1 degradation, suggesting that CaDSIZ1 is involved in drought-induced SUMOylation of CaDRHB1. CaDSIZ1 interacted with and facilitated SUMO conjugation of CaDRHB1. CaDRHB1, mainly localized in the nucleus, but also in the cytoplasm in the SUMOylation mimic state, suggesting that SUMOylation of CaDRHB1 promotes its nuclear export, leading to cytoplasmic accumulation. Moreover, CaDSIZ1-silenced pepper plants were less sensitive to ABA and considerably sensitive to drought stress, whereas CaDSIZ1-overexpressing plants displayed ABA-hypersensitive and drought-tolerant phenotypes. Collectively, our data indicate that CaDSIZ1-mediated SUMOylation of CaDRHB1 functions in ABA-mediated drought tolerance.

Inhibition of SUMO‐mediated Protein‐Protein Interactions in DNA Damage Repair

Maintenance of genomic integrity requires functional repair mechanisms. Some mechanisms include the use of sumoylation, a process in which proteins are post‐translationally modified with a small peptide SUMO (small ubiquitin‐like modifier). In response to DNA damage, multiple proteins become sumoylated. Current evidence suggests that sumoylation in this context mediates protein‐protein interactions to promote DNA damage repair. SUMO‐interacting motifs (SIMs) play an essential role in SUMO‐mediated protein‐protein interactions. The SIM on a protein is a region that allows it to recognize and bind the SUMO attached to another protein. Previous studies have shown that inhibiting this type of interaction can lead to sensitized cancer cells by impairing DNA damage repair and response. Thus, this project aims to study a similar inhibition by overexpressing SIMs naturally found in two proteins, Slx5 and Sgs1, in the eukaryotic model organism, Saccharomyces cerevisiae. Both proteins are a part of their respective protein complexes, and their SIMs serve as a functional way for the complexes to interact with sumoylated proteins during DNA repair. We hypothesize that overexpressed SIMs will disrupt endogenous SUMO‐SIM interactions important for DNA damage repair and that cells with overexpression of the SIM would be sensitized to DNA damaging agents. We observed that cells overexpressing SIMs from Slx5 exhibited a growth defect compared to cells without overexpression. However, when treated with DNA damaging agents, such as methyl methanesulfonate and UV, cells with SIM overexpression exhibited little to no sensitivity compared to the wild type.To further explore the effect of SUMO‐SIM interaction, we have designed another protein containing the DNA‐binding (SAP) domain of Siz2, a SUMO protein ligase specific to DNA damage, and the SUMO‐binding domain of Ulp1, a SUMO isopeptidase, to potentially disrupt SUMO:SIM interactions involved in DNA repair. Inclusion of the SAP domain along with a nuclear localization signal (NLS) is hypothesized to increase the nuclear localization of the protein during DNA repair, which will be observed using fluorescent microscopy of a GFP tag incorporated into the protein. With increased nuclear localization and inclusion of the SUMO‐binding domain, we hypothesize that there will be disruption to the SUMO:SIM interactions involved in DNA damage repair, resulting in an increase in the cells’ sensitivity to DNA damaging agents.

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.

Neuronal activity-induced SUMOylation of Akt1 by PIAS3 is required for long-term potentiation of synaptic transmission.

Neuronal activity regulates spatial distribution of the SUMOylation system in cytosolic and dendritic sites, which has been implicated in learning, memory, and underlying synaptic structural and functional remodeling in the hippocampus. However, the functional target proteins for activated small ubiquitin-like modifiers (SUMOs) and downstream molecular consequences behind long-term potentiation (LTP) of synaptic plasticity remain to be elucidated. In this study, we showed that N-methyl-D-aspartate receptor-mediated neuronal activity induced the covalent modification of cytosolic Akt1 by small ubiquitin-like modifier 1 (SUMO1) in rat cortical and hippocampal CA1 neurons. Protein inhibitor of activated STAT3 (PIAS3) was involved in the activity-induced Akt1 SUMO1-ylation, and K64 and K276 residues were major SUMOylated sites. Importantly, Akt1 SUMOylation at K64 and K276 enhanced its enzymatic activity and facilitated T308 phosphorylation. Furthermore, the N-terminal SAP domain of PIAS3 bound Akt1 directly. The disruption of Akt1-PIAS3 interaction by Tat-SAP, a synthetic Tat-fused cell-permeable peptide containing PIAS3 SAP domain, inhibited neuronal activity-induced Akt1 SUMOylation and impaired LTP expression and late phase LTP maintenance in the hippocampus. Correlatedly, Tat-SAP not only blocked the LTP-related extracellular signal-regulated kinase (ERK)1/2-Elk-1-brain-derived neurotrophic factor (BDNF)/Arc signaling, but also disrupted mammalian target of rapamycin (mTOR)-eIF4E-binding protein 1 (4E-BP1) pathway. These findings reveal an activity-induced Akt1 SUMOylation by PIAS3 that contributes to ERK1/2-BDNF/Arc and mTOR-4E-BP1 cascades, and in turn, long-lasting excitatory synaptic responses.

Structure specific DNA recognition by the SLX1–SLX4 endonuclease complex

The SLX1–SLX4 structure-specific endonuclease complex is involved in processing diverse DNA damage intermediates, including resolution of Holliday junctions, collapse of stalled replication forks and removal of DNA flaps. The nuclease subunit SLX1 is inactive on its own, but become activated upon binding to SLX4 via its conserved C-terminal domain (CCD). Yet, how the SLX1–SLX4 complex recognizes specific DNA structure and chooses cleavage sites remains unknown. Here we show, through a combination of structural, biochemical and computational analyses, that the SAP domain of SLX4 is critical for efficient and accurate processing of 5′-flap DNA. It binds the minor groove of DNA about one turn away from the flap junction, and the 5′-flap is implicated in binding the core domain of SLX1. This binding mode accounts for specific recognition of 5′-flap DNA and specification of cleavage site by the SLX1–SLX4 complex.

Epigenetic and Transcriptomic Profiling of MLL Rearranged Infant Acute Lymphoblastic Leukemia

[Background] Infant acute lymphoblastic leukemia (ALL) accounts for less than 5% of childhood leukemia, and is characterized by Mixed lineage leukemia (MLL also called KMT2A ) rearrangements found in approximately 80% of patients. Also found in 5-10% of ALL in older children and adults, MLL -rearrangement positivity is highly associated with poor outcome in ALL; there is an urgent need for molecular findings of potential therapeutic targets and/or risk stratification markers, which can be translated into the clinical practice. Recently, the mechanisms that MLL fusion proteins recruit epigenetic regulators, including histone H3 lysine 79 methyltransferase DOT1L and BET family histone readers, to activate the transcription of target genes have been reported, suggesting epigenetic dysregulation should play a central role in the leukemogenesis. However, DNA-level methylation profiles of MLL -rearranged leukemia and their associations with gene expression profiles are not fully characterized.[Materials and Methods] Our cohort included samples from the Infant Leukemia Sub-committee, Japan Children's Cancer Group. Both methylation array and RNA sequencing were performed on the diagnostic leukemia samples from 38 infants with ALL, consisting of 30 cases with MLL -rearrangements identified by fluorescence in situ hybridization or Southern blotting, and 8 MLL -germline cases. Methylation beta values were normalized with beta-mixture quantile normalization method. For RNA sequencing data analysis, we used publicly available Genomon Pipeline.[Results] RNA sequencing confirmed MLL -rearrangements in 30 cases, whose partner genes comprised AFF1 (n=15), MLLT1 (n=6), MLLT3 (n=3), MLLT10 (n=2), EPS15 (n=1), AFF2 (n=1), ABI1 (n=1), and SARNP (n=1). As expected, DNA methylation-based hierarchical clustering divided the cases into 2 clusters with highly distinctive methylation profiles: MLL -rearranged cluster (MLL -R cluster) and MLL -germline cluster (MLL -G cluster). Intriguingly, 3 out of 30 (10%) MLL -rearranged cases were classified into MLL -G cluster; these cases revealed uncommon partner genes, such as ABI1 , SARNP , and MLLT10, respectively. ABI1, an Eps8 SH3 domain-binding protein, and SARNP, a SAP domain containing ribonucleoprotein, are rare fusion partners of MLL. The MLL -germline leukemia-like methylation profiles suggested different leukemogenic mechanisms from common fusion partners, such as AFF1 and MLLT1, which directly or indirectly interact with DOT1L and positive transcription elongation factor b. Because DOT1L-binding domain of MLLT10 was retained in both of the 2 cases with MLL-MLLT10 in our cohort, additional mechanisms causing MLL-germline leukemia-like methylation in a single case may be present. As a result of inter-cluster comparison, MLL -R clusterexhibited global hypermethylation compared to MLL -G cluster (P -value < 2.2 × 10−16, Kolmogorov-Smirnov test). Methylation-based gene set enrichment analysis demonstrated significant promoter hypermethylation on cell development/differentiation-related gene sets and hypomethylation on DNA replication/cell proliferation-related gene sets (P -value < 0.01, hypergeometric test) in MLL -G cluster. Consistently, RNA sequencing-based gene expression analysis demonstrated overexpression of cell cycle regulators, such as cyclins/cyclin-dependent kinases, in MLL -R cluster, which may be associated with more aggressively proliferating diseases.[Conclusion] Corresponding to known aberrant histone modifications in MLL -rearranged leukemia, largely different DNA methylation profiles between MLL -R and MLL -G clusters have been demonstrated. Because DNA-level methylation on CpG dinucleotides interactively affects histone modifications, these results implicated aberrant DNA methylation in the pathogenesis of MLL -rearranged leukemia as a part of epigenetic dysregulation. We also identified a subset of MLL -rearranged cases with MLL -germline leukemia-like methylation/expression profiles, indicating that not only MLL -rearrangement positivity, but also the types of MLL -rearrangement partners may affect a biological feature of leukemic cells. These cases may possibly be therapeutically stratified as relatively lower risk group compared to other MLL -rearranged cases. Further detailed analyses using clinical information are planned. DisclosuresNo relevant conflicts of interest to declare.

TbSAP is a novel chromatin protein repressing metacyclic variant surface glycoprotein expression sites in bloodstream form Trypanosoma brucei

The African trypanosome Trypanosoma brucei is a unicellular eukaryote, which relies on a protective variant surface glycoprotein (VSG) coat for survival in the mammalian host. A single trypanosome has >2000 VSG genes and pseudogenes of which only one is expressed from one of ∼15 telomeric bloodstream form expression sites (BESs). Infectious metacyclic trypanosomes present within the tsetse fly vector also express VSG from a separate set of telomeric metacyclic ESs (MESs). All MESs are silenced in bloodstream form T. brucei. As very little is known about how this is mediated, we performed a whole genome RNAi library screen to identify MES repressors. This allowed us to identify a novel SAP domain containing DNA binding protein which we called TbSAP. TbSAP is enriched at the nuclear periphery and binds both MESs and BESs. Knockdown of TbSAP in bloodstream form trypanosomes did not result in cells becoming more ‘metacyclic-like'. Instead, there was extensive global upregulation of transcripts including MES VSGs, VSGs within the silent VSG arrays as well as genes immediately downstream of BES promoters. TbSAP therefore appears to be a novel chromatin protein playing an important role in silencing the extensive VSG repertoire of bloodstream form T. brucei.

SAP domain forms a flexible part of DNA aperture in Ku70/80.

Nonhomologous end joining (NHEJ) is a DNA repair mechanism that religates double‐strand DNA breaks to maintain genomic integrity during the entire cell cycle. The Ku70/80 complex recognizes DNA breaks and serves as an essential hub for recruitment of NHEJ components. Here, we describe intramolecular interactions of the Ku70 C‐terminal domain, known as the SAP domain. Using single‐particle cryo‐electron microscopy, mass spectrometric analysis of intermolecular cross‐linking and molecular modelling simulations, we captured variable positions of the SAP domain depending on DNA binding. The first position was localized at the DNA aperture in the Ku70/80 apo form but was not observed in the DNA‐bound state. The second position, which was observed in both apo and DNA‐bound states, was found below the DNA aperture, close to the helical arm of Ku70. The localization of the SAP domain in the DNA aperture suggests a function as a flexible entry gate for broken DNA.DatabasesEM maps have been deposited in EMDB (EMD‐11933). Coordinates have been deposited in Protein Data Bank (PDB 7AXZ). Other data are available from corresponding authors upon a request.

C-Terminal Extensions of Ku70 and Ku80 Differentially Influence DNA End Binding Properties.

The Ku70/80 heterodimer binds to DNA ends and attracts other proteins involved in the non-homologous end-joining (NHEJ) pathway of DNA double-strand break repair. We developed a novel assay to measure DNA binding and release kinetics using differences in Förster resonance energy transfer (FRET) of the ECFP-Ku70/EYFP-Ku80 heterodimer in soluble and DNA end bound states. We confirmed that the relative binding efficiencies of various DNA substrates (blunt, 3 nucleotide 5′ extension, and DNA hairpin) measured in the FRET assay reflected affinities obtained from direct measurements using surface plasmon resonance. The FRET assay was subsequently used to investigate Ku70/80 behavior in the context of a DNA-dependent kinase (DNA-PK) holocomplex. As expected, this complex was much more stable than Ku70/80 alone, and its stability was influenced by DNA-PK phosphorylation status. Interestingly, the Ku80 C-terminal extension contributed to DNA-PK complex stability but was not absolutely required for its formation. The Ku70 C-terminal SAP domain, on the other hand, was required for the stable association of Ku70/80 to DNA ends, but this effect was abrogated in DNA-PK holocomplexes. We conclude that FRET measurements can be used to determine Ku70/80 binding kinetics. The ability to do this in complex mixtures makes this assay particularly useful to study larger NHEJ protein complexes on DNA ends.

The SUMO E3 Ligase SIZ1 Negatively Regulates Shoot Regeneration.

Plants form calluses and regenerate new organs when incubated on phytohormone-containing media. While accumulating evidence suggests that these regenerative processes are governed by transcriptional networks orchestrating wound response and developmental transitions, it remains unknown if posttranslational regulatory mechanisms are involved in this process. In this study, we demonstrate that SAP AND MIZ1 DOMAIN- CONTAINING LIGASE1 (SIZ1), an E3 ligase-catalyzing attachment of the SMALL UBIQUITIN-LIKE MODIFIER (SUMO) to proteins, regulates wound-induced signal transduction and organ regeneration in Arabidopsis (Arabidopsis thaliana). We show that loss-of-function mutants for SIZ1 exhibit overproduction of shoot meristems under in vitro tissue culture conditions, while this defect is rescued in a complementation line expressing pSIZ1::SIZ1 RNA sequencing analysis revealed that siz1-2 mutants exhibit enhanced transcriptional responses to wound stress, resulting in the hyper-induction of over 400 genes immediately after wounding. Among them, we show that elevated levels of WOUND INDUCED DEDIFFERENTIATION1 (WIND1) and WIND2 contribute to the enhanced shoot regeneration observed in siz1 mutants, as expression of the dominant-negative chimeric protein WIND1-SRDX (SUPERMAN repression domain) in siz1-3 mutants partly rescues this phenotype. Although compromised SIZ1 function does not modify the transcription of genes implicated in auxin-induced callus formation and/or pluripotency acquisition, it does lead to enhanced induction of cytokinin-induced shoot meristem regulators such as WUSCHEL, promoting the formation of WUSCHEL-expressing foci in explants. This study thus suggests that SIZ1 negatively regulates shoot regeneration in part by repressing wound-induced developmental reprogramming.