The WD40-Repeat Protein-Containing Deubiquitinase Complex: Catalysis, Regulation, and Potential for Therapeutic Intervention

Ubiquitination has emerged as an essential signaling mechanism in eukaryotes. Deubiquitinases (DUBs) counteract the activities of the ubiquitination machinery and provide another level of control in cellular ubiquitination. Not surprisingly, DUBs are subjected to stringent regulations. Besides regulation by the noncatalytic domains present in the DUB sequences, DUB-interacting proteins are increasingly realized as essential regulators for DUB activity and function. This review focuses on DUBs that are associated with WD40-repeat proteins. Many human ubiquitin-specific proteases (USPs) were found to interact with WD40-repeat proteins, but little is known as to how this interaction regulates the activity and function of USPs. In recent years, significant progress has been made in understanding a prototypical WD40-repeat protein-containing DUB complex that comprises USP1 and USP1-associated factor 1 (UAF1). It has been shown that UAF1 activates USP1 through a potential active-site modulation, and the complex formation between USP1 and UAF1 is regulated by serine phosphorylation. Recently, human USPs have been recognized as a promising target class for inhibitor discovery. Small molecule inhibitors targeting several human USPs have been reported. USP1 is involved in two major DNA damage response pathways, DNA translesion synthesis and the Fanconi anemia pathway. Inhibiting the USP1/UAF1 deubiquitinase complex represents a new strategy to potentiate cancer cells to DNA-crosslinking agents and to overcome resistance that has plagued clinical cancer chemotherapy. The progress in inhibitor discovery against USPs and the WD40-repeat protein-containing USP complex will be discussed.     

The USP1-UAF1 complex interacts with RAD51AP1 to promote homologous recombination repair

USP1 deubiquitinating enzyme and its stoichiometric binding partner UAF1 play an essential role in promoting DNA homologous recombination (HR) repair in response to various types of DNA damaging agents. Deubiquitination of FANCD2 may be attributed to the key role of USP1-UAF1 complex in regulating HR repair, however whether USP1-UAF1 promotes HR repair independently of FANCD2 deubiquitination is not known. Here we show evidence that the USP1-UAF1 complex has a FANCD2-independent function in promoting HR repair. Proteomic search of UAF1-interacting proteins revealed that UAF1 associates with RAD51AP1, a RAD51-interacting protein implicated in HR repair. We show that UAF1 mediates the interaction between USP1 and RAD51AP1, and that depletion of USP1 or UAF1 led to a decreased stability of RAD51AP1. Protein interaction mapping analysis identified some key residues within RAD51AP1 required for interacting with the USP1-UAF1 complex. Cells expressing the UAF1 interaction-deficient mutant of RAD51AP1 show increased chromosomal aberrations in response to Mitomycin C treatment. Moreover, similar to the RAD51AP1 depleted cells, the cells expressing UAF1-interaction deficient RAD51AP1 display persistent RAD51 foci following DNA damage exposure, indicating that these factors regulate a later step during the HR repair. These data altogether suggest that the USP1-UAF1 complex promotes HR repair via multiple mechanisms: through FANCD2 deubiquitination, as well as by interacting with RAD51AP1.     

Distinct conformational dynamics and allosteric networks in alpha tryptophan synthase during active catalysis

Experimental observations of enzymes under active turnover conditions have brought new insight into the role of protein motions and allosteric networks in catalysis. Many of these studies characterize enzymes under dynamic chemical equilibrium conditions, in which the enzyme is actively catalyzing both the forward and reverse reactions during data acquisition. We have previously analyzed conformational dynamics and allosteric networks of the alpha subunit of tryptophan synthase under such conditions using NMR. We have proposed that this working state represents a four to one ratio of the enzyme bound with the indole‐3‐glycerol phosphate substrate (E:IGP) to the enzyme bound with the products indole and glyceraldehyde‐3‐phosphate (E:indole:G3P). Here, we analyze the inactive D60N variant to deconvolute the contributions of the substrate‐ and products‐bound states to the working state. While the D60N substitution itself induces small structural and dynamic changes, the D60N E:IGP and E:indole:G3P states cannot entirely account for the conformational dynamics and allosteric networks present in the working state. The act of chemical bond breakage and/or formation, or possibly the generation of an intermediate, may alter the structure and dynamics present in the working state. As the enzyme transitions from the substrate‐bound to the products‐bound state, millisecond conformational exchange processes are quenched and new allosteric connections are made between the alpha active site and the surface which interfaces with the beta subunit. The structural ordering of the enzyme and these new allosteric connections may be important in coordinating the channeling of the indole product into the beta subunit.     

Two nuclear localization signals in USP1 mediate nuclear import of the USP1/UAF1 complex

The human deubiquitinase USP1 plays important roles in cancer-related processes, such as the DNA damage response, and the maintenance of the undifferentiated state of osteosarcoma cells. USP1 deubiquitinase activity is critically regulated by its interaction with the WD40 repeat-containing protein UAF1. Inhibiting the function of the USP1/UAF1 complex sensitizes cancer cells to chemotherapy, suggesting that this complex is a relevant anticancer target. Intriguingly, whereas UAF1 has been reported to locate in the cytoplasm, USP1 is a nuclear protein, although the sequence motifs that mediate its nuclear import have not been functionally characterized. Here, we identify two nuclear localization signals (NLSs) in USP1 and show that these NLSs mediate the nuclear import of the USP1/UAF1 complex. Using a cellular relocation assay based on these results, we map the UAF1-binding site to a highly conserved 100 amino acid motif in USP1. Our data support a model in which USP1 and UAF1 form a complex in the cytoplasm that subsequently translocates to the nucleus through import mediated by USP1 NLSs. Importantly, our findings have practical implications for the development of USP1-directed therapies. First, the UAF1-interacting region of USP1 identified here might be targeted to disrupt the USP1/UAF1 interaction with therapeutic purposes. On the other hand, we describe a cellular relocation assay that can be easily implemented in a high throughput setting to search for drugs that may dissociate the USP1/UAF1 complex.     

Restraining CDK1–cyclin B activation: PP2A on the cUSP(7)

USP7 inhibitors are gaining momentum as a therapeutic strategy to stabilize p53 through their ability to induce MDM2 degradation. However, these inhibitors come with an unexpected p53‐independent toxicity, via an unknown mechanism. In this issue of The EMBO Journal, Galarreta et al report how inhibition of USP7 leads to re‐distribution of PP2A from cytoplasm to nucleus and an increase of deleterious CDK1‐dependent phosphorylation throughout the cell cycle, revealing a new regulatory mechanism for the progression of S‐phase cells toward mitosis to maintain genomic integrity.Subject Categories: Cell Cycle, Post-translational Modifications, Proteolysis & Proteomics

Recent work reveals untimely activation of mitotic cyclin‐dependent kinase as a molecular basis for p53‐independent cell toxicity of USP7 deubiquitinase inhibitors.

The G2‐M transition in the eukaryotic cell cycle is a critical point to ensure that cells with damaged DNA are unable to enter the mitotic phase. This checkpoint is highly regulated by a number of kinases, including ATR, ATM and WEE1, and ends upon activation of the CDK1–cyclin B1 kinase complex (Visconti et al, 2016). Since premature activation of CDK1–cyclin B1 causes replication fork collapse, DNA damage, apoptosis, and mitotic catastrophe (Szmyd et al, 2019 and references therein), restricting CDK1–cyclin B1 activity prior to mitosis is key to maintaining genomic integrity.A body of recent work has suggested that the deubiquitinase USP7 is a master regulator of genomic integrity; it is required for DNA replication in numerous ways, including indirect regulation of cyclin A2 during the S‐phase, origin firing, and replication fork progression. USP7 also regulates mitotic entry by stabilizing PLK1, another kinase which is highly active in the M phase and ensures proper alignment of chromatids prior to segregation. Notably, USP7 inhibitors have become an attractive cancer therapeutic strategy based on their ability to trigger degradation of MDM2, and thereby stabilize p53 (Valles et al, 2020). However, there is growing evidence of USP7 inhibitor‐related toxicity that is not mediated through p53 (Lecona et al, 2016; Agathanggelou et al, 2017), indicating that USP7 inhibitors impact other cellular processes. Therefore, Galarreta et al (2021) investigated the potential functional relationship between USP7 and CDK1, given the role of both factors in regulating the cell cycle.Through a series of in vitro experiments, the authors confirmed that five USP7 inhibitors induce premature mitotic kinase activity, including increased MPM2 signal (indicative of mitosis‐specific phosphorylation events) and phosphorylation of histone H3 Ser10 (H3S10P) in all cells, regardless of where they are in the cell cycle. To determine whether USP7 affects CDK1 during the cell cycle, Galarreta et al (2021) demonstrate that cell lines treated with USP7 inhibitors exhibit reduced levels of inhibitory Tyr‐15 phosphorylation on CDK1 and increased cyclin B1 presence in the nucleus, suggesting activation of the CDK1–cyclin B1 complex. Furthermore, treatment with the CDK1 inhibitor RO3306 rescues the USP7 inhibitor‐dependent increase of mitotic activity.These observations suggest that CDK1 has the potential to catalyze mitosis‐specific phosphorylation irrespective of cell cycle phase and that cells rely on USP7‐specific deubiquitination to suppress or reverse premature CDK1 activity. Surprisingly, despite the nuclear localization of cyclin B and decrease in inhibitory CDK1 Tyr‐15 phosphorylation, USP7 inhibitors failed to drive cells into mitosis. How might this be? Nuclear localization of cyclin B normally occurs just minutes before the onset of mitosis and nuclear envelope breakdown (Santos et al, 2012), yet the nucleus remains intact following USP7 inhibition. Moreover, the decrease in Tyr‐15 phosphorylation suggests the ATR‐ and WEE1‐dependent G2/M checkpoint is inactivated by USP7 inhibition. Do these data hint at the presence of an additional, unknown regulatory mechanism controlling mitotic entry independent of the G2/M checkpoint and nuclear localization of the CDK1–cyclin B complex?To determine whether CDK1 is the driver of USP7 inhibitor toxicity, Galarreta et al exposed cells to CDK1 inhibitors and observed a reduction in apoptosis. Furthermore, CDK1 inhibitors promote cell survival in cells treated with three structurally unrelated USP7 inhibitors. Finally, CDC25A‐deficient mouse embryonic stem cells, which constitutively express low levels of CDK1, resist USP7 inhibition. Together, these data suggest that the USP7 inhibitor‐dependent toxicity is the result of CDK1‐mediated cell death. The authors note that the phosphatase PP2A is an antagonist for CDK1 in addition to being a candidate USP7 substrate (Lecona et al, 2016; Wlodarchak & Xing, 2016), and thus, they turned their attention to elucidating the connection between USP7 and PP2A. Combining biochemical and immunofluorescence studies, Galarreta et al (2021) demonstrate that USP7 interacts with two subunits of PP2A, and this interaction increases in response to USP7 inhibition. Inhibiting USP7 furthermore triggers PP2A re‐localization from the cytoplasm to the nucleus and increases the phosphorylation levels of PP2A substrates, such as AKT and PRC1. DT‐061, a chemical activator of PP2A, reduces CDK1 phosphorylation events, suggesting that PP2A deregulation is a key mediator of USP7 inhibitor‐related toxicity. Using phosphoproteomics to analyze cells treated with a USP7 inhibitor or PP2A‐inhibiting okadaic acid, the authors reveal that both treatments share a significant number of altered phosphorylated targets—especially those related to mitosis, the cell cycle, and epitopes with a CDK‐dependent motif. Thus, the effects of USP7 inhibitors on CDK1 appear to be mediated through PP2A localization to the nucleus.These unexpected findings raise several questions that potentially impact the current view of cell cycle regulation. For example, how does USP7 regulate PP2A localization and is this important for reversing CDK1‐dependent phosphorylation of mitotic substrates prior to mitosis? Does PP2A accumulation in the nucleus explain the failure of USP7‐inhibited cells to enter mitosis despite cyclin B1 nuclear localization? A role for ubiquitin signaling as a regulator of CDK1 in interphase cells has not been reported, and accordingly, new investigations will be needed to unravel the mechanisms by which USP7 controls PP2A localization.Another important question that arises is whether or not CDK1 has sufficient basal activity to phosphorylate numerous mitotic proteins independent of cell cycle phase. The observation that USP7 and PP2A act to prevent the improper accumulation of CDK1‐dependent phosphorylation even in G1 phase cells suggests this to be the case. Alternatively, USP7 activity may be required to prevent abnormal pairing of CDK1 with a cyclin that is ubiquitously expressed across the cell cycle. If so, more research will be needed to uncover how ubiquitin signaling ensures CDK1 only pairs with cyclin A and cyclin B once they accumulate later in the cell cycle.Interestingly, USP7 inhibition also causes a rapid loss in DNA synthesis of S‐phase cells, prompting the authors to perform a time course experiment to decipher the order of events following treatment (i.e., does CDK1 activation precede or follow termination of DNA replication?). High‐throughput microscopy and flow cytometry analysis reveal an immediate reduction of DNA replication, an increase of CDK1 activity, and elevated DNA damage before a detectable increase in H3S10P. Long‐term exposure of USP7 inhibitors leads to DNA damage restricted only to cells with corresponding high levels of H3S10P and MPM2. Overall, these results illustrate how inhibition of USP7 activates CDK1, disrupting DNA replication and inducing DNA damage (Fig 1).Open in a separate windowFigure 1USP7 regulates CDK1In untreated cells, CDK1 is suppressed by USP7 and PP2A, and CDK1‐cyclin B is only active during the G2/M transition. In response to treatment, USP7 facilitates PP2A localization to the nucleus. This allows CDK1 to initiate premature mitotic activity throughout the cell cycle, resulting in increased DNA damage and cellular toxicity.The finding that USP7 inhibitors caused a rapid shutdown of DNA replication brings to mind the recent findings by several groups, that CDK1 activation occurs concomitantly with the S/G2 transition and that premature CDK1 activation in S‐phase terminates replication (Akopyan et al, 2014; Lemmens et al, 2018; Saldivar et al, 2018; Deng et al, 2019; Branigan et al, 2021). According to these studies, coupling of CDK1 activation to the S/G2 transition is regulated by ATR‐CHK1 signaling, a pathway activated by DNA replication to restrain CDK1 through Tyr‐15 phosphorylation. Galarreta et al''s observation that USP7 inhibition overrides ATR‐CHK1 (i.e., Tyr‐15 phosphorylation) highlights the fundamental importance of ubiquitin signaling, and potentially PP2A localization, for ensuring proper S‐to‐M progression and genome maintenance. Ultimately, the mechanistic details of Galarreta et al''s observations remain to be elucidated, and undoubtedly, their findings will inspire future investigations. Moreover, their discovery may lead to a new strategy targeting CDK1 to mitigate unwanted toxicities in the clinic.     

Non‐recombinogenic roles for Rad52 in translesion synthesis during DNA damage tolerance

DNA damage tolerance relies on homologous recombination (HR) and translesion synthesis (TLS) mechanisms to fill in the ssDNA gaps generated during passing of the replication fork over DNA lesions in the template. Whereas TLS requires specialized polymerases able to incorporate a dNTP opposite the lesion and is error‐prone, HR uses the sister chromatid and is mostly error‐free. We report that the HR protein Rad52—but not Rad51 and Rad57—acts in concert with the TLS machinery (Rad6/Rad18‐mediated PCNA ubiquitylation and polymerases Rev1/Pol ζ) to repair MMS and UV light‐induced ssDNA gaps through a non‐recombinogenic mechanism, as inferred from the different phenotypes displayed in the absence of Rad52 and Rad54 (essential for MMS‐ and UV‐induced HR); accordingly, Rad52 is required for efficient DNA damage‐induced mutagenesis. In addition, Rad52, Rad51, and Rad57, but not Rad54, facilitate Rad6/Rad18 binding to chromatin and subsequent DNA damage‐induced PCNA ubiquitylation. Therefore, Rad52 facilitates the tolerance process not only by HR but also by TLS through Rad51/Rad57‐dependent and ‐independent processes, providing a novel role for the recombination proteins in maintaining genome integrity.     

The USP1/UAF1 complex promotes double-strand break repair through homologous recombination

Protein ubiquitination plays a key role in the regulation of a variety of DNA repair mechanisms. Protein ubiquitination is controlled by the coordinate activity of ubiquitin ligases and deubiquitinating enzymes (DUBs). The deubiquitinating enzyme USP1 regulates DNA repair and the Fanconi anemia pathway through its association with its WD40 binding partner, UAF1, and through its deubiquitination of two critical DNA repair proteins, FANCD2-Ub and PCNA-Ub. To investigate the function of USP1 and UAF1, we generated USP1^−/−, UAF1^−/−/−, and USP1^−/− UAF1^−/−/− chicken DT40 cell clones. These three clones showed similar sensitivities to chemical cross-linking agents, to a topoisomerase poison, camptothecin, and to an inhibitor of poly(ADP-ribose) polymerase (PARP), indicating that the USP1/UAF1 complex is a regulator of the cellular response to DNA damage. The hypersensitivity to both camptothecin and a PARP inhibitor suggests that the USP1/UAF1 complex promotes homologous recombination (HR)-mediated double-strand break (DSB) repair. To gain insight into the mechanism of the USP1/UAF1 complex in HR, we inactivated the nonhomologous end-joining (NHEJ) pathway in UAF1-deficient cells. Disruption of NHEJ in UAF1-deficient cells restored cellular resistance to camptothecin and the PARP inhibitor. Our results indicate that the USP1/UAF1 complex promotes HR, at least in part by suppressing NHEJ.     

SCAI promotes error‐free repair of DNA interstrand crosslinks via the Fanconi anemia pathway

DNA interstrand crosslinks (ICLs) are cytotoxic lesions that threaten genome integrity. The Fanconi anemia (FA) pathway orchestrates ICL repair during DNA replication, with ubiquitylated FANCI‐FANCD2 (ID2) marking the activation step that triggers incisions on DNA to unhook the ICL. Restoration of intact DNA requires the coordinated actions of polymerase ζ (Polζ)‐mediated translesion synthesis (TLS) and homologous recombination (HR). While the proteins mediating FA pathway activation have been well characterized, the effectors regulating repair pathway choice to promote error‐free ICL resolution remain poorly defined. Here, we uncover an indispensable role of SCAI in ensuring error‐free ICL repair upon activation of the FA pathway. We show that SCAI forms a complex with Polζ and localizes to ICLs during DNA replication. SCAI‐deficient cells are exquisitely sensitive to ICL‐inducing drugs and display major hallmarks of FA gene inactivation. In the absence of SCAI, HR‐mediated ICL repair is defective, and breaks are instead re‐ligated by polymerase θ‐dependent microhomology‐mediated end‐joining, generating deletions spanning the ICL site and radial chromosomes. Our work establishes SCAI as an integral FA pathway component, acting at the interface between TLS and HR to promote error‐free ICL repair.     

Inactivation of Uaf1 Causes Defective Homologous Recombination and Early Embryonic Lethality in Mice

The deubiquitinating enzyme heterodimeric complex USP1-UAF1 regulates the Fanconi anemia (FA) DNA repair pathway. Absence of this complex leads to increased cellular levels of ubiquitinated FANCD2 (FANCD2-Ub) and ubiquitinated PCNA (PCNA-Ub). Mice deficient in the catalytic subunit of the complex, USP1, exhibit an FA-like phenotype and have a cellular deficiency in homologous-recombination (HR) repair. Here, we have characterized mice deficient in the UAF1 subunit. Uaf1^+/− mice were small at birth and exhibited reduced fertility, thus resembling Usp1^−/− mice. Unexpectedly, homozygous Uaf1^−/− embryos died at embryonic day 7.5 (E7.5). These mutant embryos were small and developmentally retarded. As expected, Uaf1 deficiency in mice led to increased levels of cellular Fancd2-Ub and Pcna-Ub. Uaf1^+/− murine embryonic fibroblasts (MEFs) exhibited profound chromosome instability, genotoxin hypersensitivity, and a significant defect in homologous-recombination repair. Moreover, Uaf1^−/− mouse embryonic stem cells (mESCs) showed chromosome instability, genotoxin hypersensitivity, and impaired Fancd2 focus assembly. Similar to USP1 knockdown, UAF1 knockdown in tumor cells caused suppression of tumor growth in vivo. Taken together, our data demonstrate the important regulatory role of the USP1-UAF1 complex in HR repair through its regulation of the FANCD2-Ub and PCNA-Ub cellular pools.     

USP7 limits CDK1 activity throughout the cell cycle

Chemical inhibitors of the deubiquitinase USP7 are currently being developed as anticancer agents based on their capacity to stabilize P53. Regardless of this activity, USP7 inhibitors also generate DNA damage in a p53‐independent manner. However, the mechanism of this genotoxicity and its contribution to the anticancer effects of USP7 inhibitors are still under debate. Here we show that, surprisingly, even if USP7 inhibitors stop DNA replication, they also induce a widespread activation of CDK1 throughout the cell cycle, which leads to DNA damage and is toxic for mammalian cells. In addition, USP7 interacts with the phosphatase PP2A and supports its active localization in the cytoplasm. Accordingly, inhibition of USP7 or PP2A triggers very similar changes of the phosphoproteome, including a widespread increase in the phosphorylation of CDK1 targets. Importantly, the toxicity of USP7 inhibitors is alleviated by lowering CDK1 activity or by chemical activation of PP2A. Our work reveals that USP7 limits CDK1 activity at all cell cycle stages, providing a novel mechanism that explains the toxicity of USP7 inhibitors through untimely activation of CDK1.     

A noncanonical cysteine protease USP1 is activated through active site modulation by USP1-associated factor 1

Ubiquitin-specific proteases (USPs) constitute the largest family of the human deubiquitinating enzymes. USP1 belongs to the cysteine protease family and contains a catalytic triad comprised of C90, H593, and D751. Notably, the catalytic activity of USP1 is stimulated through the formation of a tight complex with a WD40 repeat protein UAF1 (USP1-associated factor 1). Our kinetic analyses revealed a general base catalysis in USP1/UAF1, in contrast to an ion-pair mechanism as demonstrated for papain and cathepsin. The pK(a) value of the catalytic cysteine was determined to be 8.67 ± 0.07 in a pH-dependent inactivation study of USP1/UAF1 by iodoacetamide. A normal solvent kinetic isotope effect of 2.8 for k(cat) and 3.0 for k(cat)/K(m) was observed in the USP1/UAF1-catalyzed hydrolysis of ubiquitin-AMC substrate. Moreover, proton inventory analysis supported the transfer of a single solvent-derived proton in the transition state. Our study also revealed the molecular basis for the activation of USP1 by UAF1. Although the pK(a) of the catalytic cysteine in USP1 and USP1/UAF1 was almost identical, the pK(a) of the catalytic histidine in USP1/UAF1 was 0.43 pH unit lower than that in USP1, which facilitates general base catalysis at a neutral pH and contributes to the elevated catalytic efficiency. We ruled out that the higher catalytic efficiency is due to a tighter binding of ubiquitin. Our results support a regulatory mechanism in which UAF1 activates USP1 by modulating its active site conformation. This finding has a general implication for the regulation of USPs that form complex with partner proteins.     

PIF1 helicase promotes break‐induced replication in mammalian cells

Break‐induced replication (BIR) is a specialized homologous‐recombination pathway for DNA double‐strand break (DSB) repair, which often induces genome instability. In this study, we establish EGFP‐based recombination reporters to systematically study BIR in mammalian cells and demonstrate an important role of human PIF1 helicase in promoting BIR. We show that at endonuclease cleavage sites, PIF1‐dependent BIR is used for homology‐initiated recombination requiring long track DNA synthesis, but not short track gene conversion (STGC). We also show that structure formation‐prone AT‐rich DNA sequences derived from common fragile sites (CFS‐ATs) induce BIR upon replication stress and oncogenic stress, and PCNA‐dependent loading of PIF1 onto collapsed/broken forks is critical for BIR activation. At broken replication forks, even STGC‐mediated repair of double‐ended DSBs depends on POLD3 and PIF1, revealing an unexpected mechanism of BIR activation upon replication stress that differs from the conventional BIR activation model requiring DSB end sensing at endonuclease‐generated breaks. Furthermore, loss of PIF1 is synthetically lethal with loss of FANCM, which is involved in protecting CFS‐ATs. The breast cancer‐associated PIF1 mutant L319P is defective in BIR, suggesting a direct link of BIR to oncogenic processes.     

Centrosome defects cause microcephaly by activating the 53BP1‐USP28‐TP53 mitotic surveillance pathway

Mutations in centrosome genes deplete neural progenitor cells (NPCs) during brain development, causing microcephaly. While NPC attrition is linked to TP53‐mediated cell death in several microcephaly models, how TP53 is activated remains unclear. In cultured cells, mitotic delays resulting from centrosome loss prevent the growth of unfit daughter cells by activating a pathway involving 53BP1, USP28, and TP53, termed the mitotic surveillance pathway. Whether this pathway is active in the developing brain is unknown. Here, we show that the depletion of centrosome proteins in NPCs prolongs mitosis and increases TP53‐mediated apoptosis. Cell death after a delayed mitosis was rescued by inactivation of the mitotic surveillance pathway. Moreover, 53BP1 or USP28 deletion restored NPC proliferation and brain size without correcting the upstream centrosome defects or extended mitosis. By contrast, microcephaly caused by the loss of the non‐centrosomal protein SMC5 is also TP53‐dependent but is not rescued by loss of 53BP1 or USP28. Thus, we propose that mutations in centrosome genes cause microcephaly by delaying mitosis and pathologically activating the mitotic surveillance pathway in the developing brain.     

Deubiquitination of FANCD2 is required for DNA crosslink repair

Monoubiquitination of FANCD2 and PCNA promotes DNA repair. It causes chromatin accumulation of FANCD2 and facilitates PCNA's recruitment of translesion polymerases to stalled replication. USP1, a protease that removes monoubiquitin from FANCD2 and PCNA, was thought to reverse the DNA damage response of these substrates. We disrupted USP1 in chicken cells to dissect its role in a stable genetic system. USP1 ablation increases FANCD2 and PCNA monoubiquitination but unexpectedly results in DNA crosslinker sensitivity. This defective DNA repair is associated with constitutively chromatin-bound, monoubiquitinated FANCD2. In contrast, persistent PCNA monoubiquitination has negligible impact on DNA repair or mutagenesis. USP1 was previously shown to autocleave after DNA damage. In DT40, USP1 autocleavage is not stimulated by DNA damage, and expressing a noncleavable mutant in the USP1 knockout strain partially rescues crosslinker sensitivity. We conclude that efficient DNA crosslink repair requires FANCD2 deubiquitination, whereas FANCD2 monoubiquitination is not dependent on USP1 autocleavage.     

The CRL4DCAF1 cullin‐RING ubiquitin ligase is activated following a switch in oligomerization state

The cullin‐4‐based RING‐type (CRL4) family of E3 ubiquitin ligases functions together with dedicated substrate receptors. Out of the ˜29 CRL4 substrate receptors reported, the DDB1‐ and CUL4‐associated factor 1 (DCAF1) is essential for cellular survival and growth, and its deregulation has been implicated in tumorigenesis. We carried out biochemical and structural studies to examine the structure and mechanism of the CRL4^DCAF1 ligase. In the 8.4 Å cryo‐EM map of CRL4^DCAF1, four CUL4‐RBX1‐DDB1‐DCAF1 protomers are organized into two dimeric sub‐assemblies. In this arrangement, the WD40 domain of DCAF1 mediates binding with the cullin C‐terminal domain (CTD) and the RBX1 subunit of a neighboring CRL4^DCAF1 protomer. This renders RBX1, the catalytic subunit of the ligase, inaccessible to the E2 ubiquitin‐conjugating enzymes. Upon CRL4^DCAF1 activation by neddylation, the interaction between the cullin CTD and the neighboring DCAF1 protomer is broken, and the complex assumes an active dimeric conformation. Accordingly, a tetramerization‐deficient CRL4^DCAF1 mutant has higher ubiquitin ligase activity compared to the wild‐type. This study identifies a novel mechanism by which unneddylated and substrate‐free CUL4 ligases can be maintained in an inactive state.     

Phosphoproteomics reveals a distinctive Mec1/ATR signaling response upon DNA end hyper‐resection

The Mec1/ATR kinase is crucial for genome maintenance in response to a range of genotoxic insults, but it remains unclear how it promotes context‐dependent signaling and DNA repair. Using phosphoproteomic analyses, we uncovered a distinctive Mec1/ATR signaling response triggered by extensive nucleolytic processing (resection) of DNA ends. Budding yeast cells lacking Rad9, a checkpoint adaptor and an inhibitor of resection, exhibit a selective increase in Mec1‐dependent phosphorylation of proteins associated with single‐strand DNA (ssDNA) transactions, including the ssDNA‐binding protein Rfa2, the translocase/ubiquitin ligase Uls1, and the Sgs1‐Top3‐Rmi1 (STR) complex that regulates homologous recombination (HR). Extensive Mec1‐dependent phosphorylation of the STR complex, mostly on the Sgs1 helicase subunit, promotes an interaction between STR and the DNA repair scaffolding protein Dpb11. Fusion of Sgs1 to phosphopeptide‐binding domains of Dpb11 strongly impairs HR‐mediated repair, supporting a model whereby Mec1 signaling regulates STR upon hyper‐resection to influence recombination outcomes. Overall, the identification of a distinct Mec1 signaling response triggered by hyper‐resection highlights the multi‐faceted action of this kinase in the coordination of checkpoint signaling and HR‐mediated DNA repair.     

Uniform binding and negative catalysis at the origin of enzymes

Enzymes are well known for their catalytic abilities, some even reaching “catalytic perfection” in the sense that the reaction they catalyze has reached the physical bound of the diffusion rate. However, our growing understanding of enzyme superfamilies has revealed that only some share a catalytic chemistry while others share a substrate‐handle binding motif, for example, for a particular phosphate group. This suggests that some families emerged through a “substrate‐handle‐binding‐first” mechanism (“binding‐first” for brevity) instead of “chemistry‐first” and we are, therefore, left to wonder what the role of non‐catalytic binders might have been during enzyme evolution. In the last of their eight seminal, back‐to‐back articles from 1976, John Albery and Jeremy Knowles addressed the question of enzyme evolution by arguing that the simplest mode of enzyme evolution is what they defined as “uniform binding” (parallel stabilization of all enzyme‐bound states to the same degree). Indeed, we show that a uniform‐binding proto‐catalyst can accelerate a reaction, but only when catalysis is already present, that is, when the transition state is already stabilized to some degree. Thus, we sought an alternative explanation for the cases where substrate‐handle‐binding preceded any involvement of a catalyst. We find that evolutionary starting points that exhibit negative catalysis can redirect the reaction''s course to a preferred product without need for rate acceleration or product release; that is, if they do not stabilize, or even destabilize, the transition state corresponding to an undesired product. Such a mechanism might explain the emergence of “binding‐first” enzyme families like the aldolase superfamily.