N-degron pathways.

An N-degron is a degradation signal whose main determinant is a "destabilizing" N-terminal residue of a protein. Specific N-degrons, discovered in 1986, were the first identified degradation signals in short-lived intracellular proteins. These N-degrons are recognized by a ubiquitin-dependent proteolytic system called the Arg/N-degron pathway. Although bacteria lack the ubiquitin system, they also have N-degron pathways. Studies after 1986 have shown that all 20 amino acids of the genetic code can act, in specific sequence contexts, as destabilizing N-terminal residues. Eukaryotic proteins are targeted for the conditional or constitutive degradation by at least five N-degron systems that differ both functionally and mechanistically: the Arg/N-degron pathway, the Ac/N-degron pathway, the Pro/N-degron pathway, the fMet/N-degron pathway, and the newly named, in this perspective, GASTC/N-degron pathway (GASTC = Gly, Ala, Ser, Thr, Cys). I discuss these systems and the expanded terminology that now encompasses the entire gamut of known N-degron pathways.

Establishment and characterization of mouse lines useful for endogenous protein degradation via an improved auxin-inducible degron system (AID2).

The development of new technologies opens new avenues in the research field. Gene knockout is a key method for analyzing gene function in mice. Currently, conditional gene knockout strategies are employed to examine temporal and spatial gene function. However, phenotypes are sometimes not observed because of the time required for depletion due to the long half-life of the target proteins. Protein knockdown using an improved auxin-inducible degron system, AID2, overcomes such difficulties owing to rapid and efficient target depletion. We observed depletion of AID-tagged proteins within a few to several hours by a simple intraperitoneal injection of the auxin analog, 5-Ph-IAA, which is much shorter than the time required for target depletion using conditional gene knockout. Importantly, the loss of protein is reversible, making protein knockdown useful to measure the effects of transient loss of protein function. Here, we also established several mouse lines useful for AID2-medicated protein knockdown, which include knock-in mouse lines in the ROSA26 locus; one expresses TIR1(F74G), and the other is the reporter expressing AID-mCherry. We also established a germ-cell-specific TIR1 line and confirmed the protein knockdown specificity. In addition, we introduced an AID tag to an endogenous protein, DCP2 via the CAS9-mediated gene editing method. We confirmed that the protein was effectively eliminated by TIR1(F74G), which resulted in the similar phenotype observed in knockout mouse within 20 h.

Fluorescent tagging of endogenous IRS2 with an auxin-dependent degron to assess dynamic intracellular localization and function.

Insulin Receptor Substrate 2 (IRS2) is a signaling adaptor protein for the insulin (IR) and Insulin-like Growth Factor-1 (IGF-1R) receptors. In breast cancer, IRS2 contributes to both initiation of primary tumor growth and establishment of secondary metastases through regulation of cancer stem cell (CSC) function and invasion. However, how IRS2 mediates its diverse functions is not well understood. We used CRISPR/Cas9-mediated gene editing to modify endogenous IRS2 to study the expression, localization, and function of this adaptor protein. A cassette containing an auxin inducible degradation (AID) sequence, 3X-FLAG tag and mNeon-green was introduced at the N-terminus of the IRS2 gene to provide rapid and reversible control of IRS2 protein degradation and analysis of endogenous IRS2 expression and localization. Live fluorescence imaging of these cells revealed that IRS2 shuttles between the cytoplasm and nucleus in response to growth regulatory signals in a PI3K-dependent manner. Inhibition of nuclear export or deletion of a putative nuclear export sequence in the C-terminal tail promotes nuclear retention of IRS2, implicating nuclear export in the mechanism by which IRS2 intracellular localization is regulated. Moreover, the acute induction of IRS2 degradation reduces tumor cell invasion, demonstrating the potential for therapeutic targeting of this adaptor protein. Our data highlight the value of our model of endogenously tagged IRS2 as a tool to study IRS2 localization and function.

A novel mutation in SoIAA20 confers cross-resistance to 2,4-Dichlorophenoxyacetic acid and other auxinic herbicides in Sonchus oleraceus.

BACKGROUND: 2,4-Dichlorophenoxyacetic acid (2,4-D) and other auxinic herbicides are important for weed control in cropping systems globally. Weeds with resistance to 2,4-D and other auxinic herbicides have evolved, including several populations of Sonchus oleraceus from multiple sites in Australia. We report the underlying mechanism in these populations that gives rise to auxinic herbicide resistance. RESULTS: We studied a total of three susceptible and eight resistant Sonchus oleraceus populations. All resistant populations had a deletion of three amino acids flanking the degron sequence of an Aux/IAA gene, SoIAA20, which was not found in the three susceptible populations. The eight populations with the resistant allele were also resistant to dicamba, fluroxypyr and clopyralid. The resistant plants also had reduced movement of 2,4-D out of the treated tissues compared to susceptible plants. CONCLUSION: The paired deletion flanking the degron region of SoIAA20 likely provides resistance to 2,4-D by restricting the movement of 2,4-D from the treated tissue to the rest of the plant. We hypothesise that this deletion keeps the 2,4-D bound to the target site. © 2024 The Author(s). Pest Management Science published by John Wiley & Sons Ltd on behalf of Society of Chemical Industry.

The ubiquitin-proteasome system regulates the formation of specialized ribosomes during high salt stress in yeast.

Rps26-deficient ribosomes are a physiologically relevant ribosome population which arises during osmotic stress to support the translation of mRNAs involved in the response to high salt in yeast. They are formed by binding of the chaperone Tsr2 to fully assembled ribosomes to release Rps26 when intracellular Na+ concentrations rise. Tsr2-mediated Rps26 release is reversible, enabling a rapid response that conserves ribosomes. However, because the concentration of Tsr2 relative to ribosomes is low, how the released Rps26•Tsr2 complex is managed to allow for accumulation of Rps26-deficient ribosomes to nearly 50% of all ribosomes remains unclear. Here we show that released Rps26 is degraded via the Pro/N-degron pathway, enabling the accumulation of Rps26-deficient ribosomes. Substitution of the N-terminal proline of Rps26 to serine increases the stability of free Rps26, limits the accumulation of Rps26-deficient ribosomes and renders yeast sensitive to high salt. The GID-complex, an E3 ubiquitin ligase, and its adaptor Gid4, mediate polyubiquitination of Rps26 at Lys66 and Lys70. Moreover, this ubiquitination event is required for Rps26 degradation, the accumulation of Rps26-deficient ribosomes and the high salt stress resistance. Together, the data show that targeted degradation of released Rps26 from the Rps26•Tsr2 complex allows Tsr2 to be recycled, thus facilitating multiple rounds of Rps26 release.

Combination of AID2 and BromoTag expands the utility of degron-based protein knockdowns.

Acute protein knockdown is a powerful approach to dissecting protein function in dynamic cellular processes. We previously reported an improved auxin-inducible degron system, AID2, but recently noted that its ability to induce degradation of some essential replication factors, such as ORC1 and CDC6, was not enough to induce lethality. Here, we present combinational degron technologies to control two proteins or enhance target depletion. For this purpose, we initially compare PROTAC-based degrons, dTAG and BromoTag, with AID2 to reveal their key features and then demonstrate control of cohesin and condensin with AID2 and BromoTag, respectively. We develop a double-degron system with AID2 and BromoTag to enhance target depletion and accelerate depletion kinetics and demonstrate that both ORC1 and CDC6 are pivotal for MCM loading. Finally, we show that co-depletion of ORC1 and CDC6 by the double-degron system completely suppresses DNA replication, and the cells enter mitosis with single-chromatid chromosomes, indicating that DNA replication is uncoupled from cell cycle control. Our combinational degron technologies will expand the application scope for functional analyses.

The enzymatic oxygen sensor cysteamine dioxygenase binds its protein substrates through their N-termini.

The non-heme iron-dependent dioxygenase 2-aminoethanethiol (aka cysteamine) dioxygenase (ADO) has recently been identified as an enzymatic oxygen sensor that coordinates cellular changes to hypoxia by regulating the stability of proteins bearing an N-terminal cysteine (Nt-cys) through the N-degron pathway. It catalyzes O2-dependent Nt-cys sulfinylation, which promotes proteasomal degradation of the target. Only a few ADO substrates have been verified, including regulators of G-protein signaling (RGS) 4 and 5, and the proinflammatory cytokine interleukin-32, all of which exhibit cell and/or tissue specific expression patterns. ADO, in contrast, is ubiquitously expressed, suggesting it can regulate the stability of additional Nt-cys proteins in an O2-dependent manner. However, the role of individual chemical groups, active site metal, amino acid composition, and globular structure on protein substrate association remains elusive. To help identify new targets and examine the underlying biochemistry of the system, we conducted a series of biophysical experiments to investigate the binding requirements of established ADO substrates RGS5 and interleukin-32. We demonstrate, using surface plasmon response and enzyme assays, that a free, unmodified Nt-thiol and Nt-amine are vital for substrate engagement through active site metal coordination, with residues next to Nt-cys moderately impacting association and catalytic efficiency. Additionally, we show, through 1H-15N heteronuclear single quantum coherence nuclear magnetic resonance titrations, that the globular portion of RGS5 has limited impact on ADO association, with interactions restricted to the N-terminus. This work establishes key features involved in ADO substrate binding, which will help identify new protein targets and, subsequently, elucidate its role in hypoxic adaptation.

Hypoxia represses pattern-triggered immune responses in Arabidopsis.

Biotic and abiotic stresses frequently co-occur in nature, yet relatively little is known about how plants co-ordinate the response to combined stresses. Protein degradation by the ubiquitin/proteasome system is central to the regulation of multiple independent stress response pathways in plants. The Arg/N-degron pathway is a subset of the ubiquitin/proteasome system that targets proteins based on their N termini and has been specifically implicated in the responses to biotic and abiotic stresses, including hypoxia, via accumulation of group VII ETHYLENE RESPONSE FACTOR (ERF-VII) transcription factors that orchestrate the onset of the hypoxia response program. Here, we investigated the role of the Arabidopsis (Arabidopsis thaliana) Arg/N-degron pathway in mediating the crosstalk between combined abiotic and biotic stresses using hypoxia treatments and the flg22 elicitor of pattern-triggered immunity (PTI), respectively. We uncovered a link between the plant transcriptional responses to hypoxia and flg22. Combined hypoxia and flg22 treatments showed that hypoxia represses the flg22 transcriptional program, as well as the expression of pattern recognition receptors, mitogen-activated protein kinase (MAPK) signalling and callose deposition during PTI through mechanisms that are mostly independent from the ERF-VIIs. These findings improve our understanding of the trade-offs between plant responses to combined abiotic and biotic stresses in the context of our efforts to increase crop resilience to global climate change. Our results also show that the well-known repressive effect of hypoxia on innate immunity in animals also applies to plants.

Nucleolin acute degradation reveals novel functions in cell cycle progression and division in TNBC.

Nucleoli are large nuclear sub-compartments where vital processes, such as ribosome assembly, take place. Technical obstacles still limit our understanding of the biological functions of nucleolar proteins in cell homeostasis and cancer pathogenesis. Since most nucleolar proteins are essential, their abrogation cannot be achieved through conventional approaches. Additionally, the biological activities of many nucleolar proteins are connected to their physiological concentration. Thus, artificial overexpression might not fully recapitulate their endogenous functions. Proteolysis-based approaches, such as the Auxin Inducible Degron (AID) system paired with CRISPR/Cas9 knock-in gene-editing, have the potential to overcome these limitations, providing unprecedented characterization of the biological activities of endogenous nucleolar proteins. We applied this system to endogenous nucleolin (NCL), one of the most abundant nucleolar proteins, and characterized the impact of its acute depletion on Triple-Negative Breast Cancer (TNBC) cell behavior. Abrogation of endogenous NCL reduced proliferation and caused defective cytokinesis, resulting in bi-nucleated tetraploid cells. Bioinformatic analysis of patient data, and quantitative proteomics using our experimental NCL-depleted model, indicated that NCL levels are correlated with the abundance of proteins involved in chromosomal segregation. In conjunction with its effects on sister chromatid dynamics, NCL abrogation enhanced the anti-proliferative effects of chemical inhibitors of mitotic modulators such as the Anaphase Promoting Complex. In summary, using the AID system in combination with CRISPR/Cas9 for endogenous gene editing, our findings indicate a novel role for NCL in supporting the completion of the cell division in TNBC models, and that its abrogation could enhance the therapeutic activity of mitotic-progression inhibitors.

Developing Device of Death Operation (DODO) to Detect Apoptosis in 2D and 3D Cultures.

The real-time detection of intracellular biological processes by encoded sensors has broad application prospects. Here, we developed a degron-based modular reporting system, the Device of Death Operation (DODO), that can monitor various biological processes. The DODO system consists of a "reporter", an "inductor", and a "degron". After zymogen activation and cleavage, the degron will be released from the "reporter", which eventually leads to the stabilization of the "reporter", and can be detected. By replacing different "inductors" and "reporters", a series of biological processes can be reported through various signals. The system can effectively report the existence of TEV protease. To prove this concept, we successfully applied the DODO system to report apoptosis in 2D and 3D cultures. In addition, the reporter based on degron will help to design protease reporters other than caspase.

A Single-step Generation of AlissAID-based Conditional Knockdown Strains Using Nanobody that Targets GFP or mCherry in Budding Yeast.

The Auxin-inducible degron (AID) system is a genetic tool that induces rapid target protein depletion in an auxin-dependent manner. Recently, two advanced AID systems-the super-sensitive AID and AID 2-were developed using an improved pair of synthetic auxins and mutated TIR1 proteins. In these AID systems, a nanomolar concentration of synthetic auxins is sufficient as a degradation inducer for target proteins. However, despite these advancements, AID systems still require the fusion of an AID tag to the target protein for degradation, potentially affecting its function and stability. To address this limitation, we developed an affinity linker-based super-sensitive AID (AlissAID) system using a single peptide antibody known as a nanobody. In this system, the degradation of GFP- or mCherry-tagged target proteins is induced in a synthetic auxin (5-Ad-IAA)-dependent manner. Here, we introduce a simple method for generating AlissAID strains targeting GFP or mCherry fusion proteins in budding yeasts. Key features • AlissAID system enables efficient degradation of the GFP or mCherry fusion proteins in a 5-Ad-IAA-depending manner. • Transforming the pAlissAID plasmids into strains with GFP- or mCherry- tagged proteins.

Fine-Tuning the Epigenetic Landscape: Chemical Modulation of Epigenome Editors.

Epigenome editing has emerged as a powerful technique for targeted manipulation of the chromatin and transcriptional landscape, employing designer DNA binding domains fused with effector domains, known as epi-editors. However, the constitutive expression of dCas9-based epi-editors presents challenges, including off-target activity and lack of temporal resolution. Recent advancements of dCas9-based epi-editors have addressed these limitations by introducing innovative switch systems that enable temporal control of their activity. These systems allow precise modulation of gene expression over time and offer a means to deactivate epi-editors, thereby reducing off-target effects associated with prolonged expression. The development of novel dCas9 effectors regulated by exogenous chemical signals has revolutionized temporal control in epigenome editing, significantly expanding the researcher's toolbox. Here, we provide a comprehensive review of the current state of these cutting-edge systems and specifically discuss their advantages and limitations, offering context to better understand their capabilities.

A small-molecule degron with a phenylpropionic acid scaffold.

Targeted protein degradation (TPD), employing proteolysis-targeting chimeras (PROTACs) composed of ligands for both a target protein and ubiquitin ligase (E3) to redirect the ubiquitin-proteasome system (UPS) to the target protein, has emerged as a promising strategy in drug discovery. However, despite the vast number of E3 ligases, the repertoire of E3 ligands utilized in PROTACs remains limited. Here, we report the discovery of a small-molecule degron with a phenylpropionic acid skeleton, derived from a known ligand of S-phase kinase-interacting protein 2 (Skp2), an E3 ligase. We used this degron to design PROTACs inducing proteasomal degradation of HaloTag-fused proteins, and identified key structural relationships. Surprisingly, our mechanistic studies excluded the involvement of Skp2, suggesting that this degron recruits other protein(s) within the UPS.

How kelch domain-containing protein 3 distinguishes between the C-end degron of herpesviral protein UL49.5 and its mutants - Insights from molecular dynamics.

The C-terminal residues of proteins can function as degrons recognized by ubiquitin ligases for proteasomal degradation. Kelch domain-containing protein 3 (KLHDC3) is a substrate receptor for E3 ubiquitin ligase (Cullin2-RING ligase) that targets the C-terminal degrons. UL49.5 is 96 amino-acid type 1 transmembrane protein from bovine herpesvirus 1. Herpesviruses have evolved highly effective strategies to evade the antiviral immune response. One of these strategies is inhibition of the antigen processing and presentation pathway by MHC I, thereby reducing the presentation of the antigenic peptides on the surface of the infected cell. Recently, it has been demonstrated that UL49.5 triggers TAP degradation via recruiting the E3 ubiquitin ligase to TAP. Moreover, the mutagenesis revealed that the mutations within the UL49.5 C-degron sequence (93RGRG96) affect binding of UL49.5 to KLHDC3. In this work the molecular dynamics of KLHDC3 in complexes with the C-terminal decapeptide of the herpesviral protein UL4.95 and its three mutants has been employed to provide a framework for understanding molecular recognition of UL49.5 by KLHDC3. The findings of this study give insights into the interactions of the various degrons with KLHDC3. During the molecular dynamics, an active RGKG mutant adopts a conformation similar to that of the wild type decapeptide, whereas the conformations of two inactive mutants, KGRG and RGRD are significantly different. Both R93K and G96D mutations impair the interactions of the C-terminal glycine with KLHDC3. The findings of this study expand the existing knowledge about the mechanism of protein recognition by Cullin2-RING ligases thus contributing to the design of antiviral and anticancer drugs that can selectively promote or inhibit degradation of the proteins of interest.

UPS-dependent strategies of protein quality control degradation.

The degradation of damaged proteins is critical for tissue integrity and organismal health because damaged proteins have a high propensity to form aggregates. E3 ubiquitin ligases are key regulators of protein quality control (PQC) and mediate the selective degradation of damaged proteins, a process termed 'PQC degradation' (PQCD). The degradation signals (degrons) that trigger PQCD are based on hydrophobic sites that are normally buried within the native protein structure. However, an open question is how PQCD-specialized E3 ligases distinguish between transiently misfolded proteins, which can be efficiently refolded, and permanently damaged proteins, which must be degraded. While significant progress has been made in characterizing degradation determinants, understanding the key regulatory signals of cellular and organismal PQCD pathways remains a challenge.

A temporal sequence of heterochronic gene activities promotes stage-specific developmental events in Caenorhabditis elegans.

The heterochronic genes of the nematode Caenorhabditis elegans control the succession of postembryonic developmental events. The 4 core heterochronic genes lin-14, lin-28, hbl-1, and lin-41 act in a sequence to specify cell fates specific to each of the 4 larval stages. It was previously shown that lin-14 has 2 activities separated in time that promote L1 and L2 developmental events, respectively. Using the auxin-inducible degron system, we find that lin-28 and hbl-1 each have 2 activities that control L2 and L3 events which are also separated in time. Relative to events they control, both lin-28 and hbl-1 appear to act just prior to or concurrently with events of the L2. Relative to each other, lin-28 and hbl-1 appear to act simultaneously. By contrast, the lin-14 activity controlling L2 events precedes those of lin-28 and hbl-1 controlling the same events, suggesting that lin-14's regulation of lin-28 is responsible for the delay. Likewise, the activities of lin-28 and hbl-1 controlling L3 fates act well in advance of those fates, suggesting a similar regulatory gap. lin-41 acts early in the L3 to affect fates of the L4, although it was not possible to determine whether it too has 2 temporally separated activities. We also uncovered a feedback phenomenon that prevents the reactivation of heterochronic gene activity late in development after it has been downregulated. This study places the heterochronic gene activities into a timeline of postembryonic development relative to one another and to the developmental events whose timing they control.

Structural study for substrate recognition of human N-terminal glutamine amidohydrolase 1 in the arginine N-degron pathway.

The N-degron pathway determines the half-life of proteins by selectively destabilizing the proteins bearing N-degrons. N-terminal glutamine amidohydrolase 1 (NTAQ1) plays an essential role in the arginine N-degron (Arg/N-degron) pathway as an initializing enzyme via the deamidation of the N-terminal (Nt) glutamine (Gln). However, the Nt-serine-bound conformation of hNTAQ1 according to the previously identified crystal structure suggests the possibility of other factors influencing the recognition of Nt residues by hNTAQ1. Hence, in the current study, we aimed to further elucidate the substrate recognition of hNTAQ1; specifically, we explored 12 different substrate-binding conformations of hNTAQ1 depending on the subsequent residue of Nt-Gln. Results revealed that hNTAQ1 primarily interacts with the protein Nt backbone, instead of the side chain, for substrate recognition. Here, we report that the Nt backbone of proteins appears to be a key component of hNTAQ1 function and is the main determinant of substrate recognition. Moreover, not all second residues from Nt-Gln, but rather distinctive and charged residues, appeared to aid in detecting substrate recognition. These new findings define the substrate-recognition process of hNTAQ1 and emphasize the importance of the subsequent Gln residue in the Nt-Gln degradation system. Our extensive structural and biochemical analyses provide insights into the substrate specificity of the N-degron pathway and shed light on the mechanism underlying hNTAQ1 substrate recognition. An improved understanding of the protein degradation machinery could aid in developing therapies to promote overall health through enhanced protein regulation, such as targeted protein therapies.

Pros and cons of auxin-inducible degron as a tool for regulated depletion of telomeric proteins from Saccharomyces cerevisiae.

To assess the immediate responses of the yeast cells to telomere defects, we employed the auxin-inducible degron (AID) enabling rapid depletion of essential (Rap1, Tbf1, Cdc13, Stn1) and non-essential (Est1, Est2, Est3) telomeric proteins. Using two variants of AID systems, we show that most of the studied proteins are depleted within 10-30 min after the addition of auxin. As expected, depletion of essential proteins yields nondividing cells, provided that the strains are cultivated in an appropriate carbon source and at temperatures lower than 28°C. Cells with depleted Cdc13 and Stn1 exhibit extension of the single-stranded overhang as early as 3 h after addition of auxin. Notably, prolonged incubation of strains carrying AID-tagged essential proteins in the presence of auxin resulted in the appearance of auxin-resistant clones, caused at least in part by mutations within the OsTIR1 gene. Upon assessing the length of telomeres in strains carrying AID-tagged non-essential telomeric proteins, we found that the depletion of Est1 and Est3 leads to auxin-dependent telomere shortening. However, the EST3-AID strain had slightly shorter telomeres even in the absence of auxin. Furthermore, a strain with the AID-tagged version of Est2 (catalytic subunit of telomerase) not only had shorter telomeres in the absence of auxin but also did not exhibit auxin-dependent telomere shortening. Our results demonstrate that while AID can be useful in assessing immediate cellular responses to telomere deprotection, each strain must be carefully evaluated for the effect of AID-tag on the properties of the protein of interest.

Non-canonical substrate recognition by the human WDR26-CTLH E3 ligase regulates prodrug metabolism.

The yeast glucose-induced degradation-deficient (GID) E3 ubiquitin ligase forms a suite of complexes with interchangeable receptors that selectively recruit N-terminal degron motifs of metabolic enzyme substrates. The orthologous higher eukaryotic C-terminal to LisH (CTLH) E3 complex has been proposed to also recognize substrates through an alternative subunit, WDR26, which promotes the formation of supramolecular CTLH E3 assemblies. Here, we discover that human WDR26 binds the metabolic enzyme nicotinamide/nicotinic-acid-mononucleotide-adenylyltransferase 1 (NMNAT1) and mediates its CTLH E3-dependent ubiquitylation independently of canonical GID/CTLH E3-family substrate receptors. The CTLH subunit YPEL5 inhibits NMNAT1 ubiquitylation and cellular turnover by WDR26-CTLH E3, thereby affecting NMNAT1-mediated metabolic activation and cytotoxicity of the prodrug tiazofurin. Cryoelectron microscopy (cryo-EM) structures of NMNAT1- and YPEL5-bound WDR26-CTLH E3 complexes reveal an internal basic degron motif of NMNAT1 essential for targeting by WDR26-CTLH E3 and degron mimicry by YPEL5's N terminus antagonizing substrate binding. Thus, our data provide a mechanistic understanding of how YPEL5-WDR26-CTLH E3 acts as a modulator of NMNAT1-dependent metabolism.

The bacterial DNA sliding clamp, ß-clamp: structure, interactions, dynamics and drug discovery.

DNA replication is a tightly coordinated event carried out by a multiprotein replication complex. An essential factor in the bacterial replication complex is the ring-shaped DNA sliding clamp, ß-clamp, ensuring processive DNA replication and DNA repair through tethering of polymerases and DNA repair proteins to DNA. ß -clamp is a hub protein with multiple interaction partners all binding through a conserved clamp binding sequence motif. Due to its central role as a DNA scaffold protein, ß-clamp is an interesting target for antimicrobial drugs, yet little effort has been put into understanding the functional interactions of ß-clamp. In this review, we scrutinize the ß-clamp structure and dynamics, examine how its interactions with a plethora of binding partners are regulated through short linear binding motifs and discuss how contexts play into selection. We describe the dynamic process of clamp loading onto DNA and cover the recent advances in drug development targeting ß-clamp. Despite decades of research in ß-clamps and recent landmark structural insight, much remains undisclosed fostering an increased focus on this very central protein.