Cullin-RING ubiquitin E3 ligase regulation by the COP9 signalosome.

The cullin-RING ubiquitin E3 ligase (CRL) family comprises over 200 members in humans. The COP9 signalosome complex (CSN) regulates CRLs by removing their ubiquitin-like activator NEDD8. The CUL4A-RBX1-DDB1-DDB2 complex (CRL4A(DDB2)) monitors the genome for ultraviolet-light-induced DNA damage. CRL4A(DBB2) is inactive in the absence of damaged DNA and requires CSN to regulate the repair process. The structural basis of CSN binding to CRL4A(DDB2) and the principles of CSN activation are poorly understood. Here we present cryo-electron microscopy structures for CSN in complex with neddylated CRL4A ligases to 6.4 Å resolution. The CSN conformers defined by cryo-electron microscopy and a novel apo-CSN crystal structure indicate an induced-fit mechanism that drives CSN activation by neddylated CRLs. We find that CSN and a substrate cannot bind simultaneously to CRL4A, favouring a deneddylated, inactive state for substrate-free CRL4 complexes. These architectural and regulatory principles appear conserved across CRL families, allowing global regulation by CSN.

Crystal structure of the human COP9 signalosome.

Ubiquitination is a crucial cellular signalling process, and is controlled on multiple levels. Cullin-RING E3 ubiquitin ligases (CRLs) are regulated by the eight-subunit COP9 signalosome (CSN). CSN inactivates CRLs by removing their covalently attached activator, NEDD8. NEDD8 cleavage by CSN is catalysed by CSN5, a Zn(2+)-dependent isopeptidase that is inactive in isolation. Here we present the crystal structure of the entire ∼350-kDa human CSN holoenzyme at 3.8 Å resolution, detailing the molecular architecture of the complex. CSN has two organizational centres: a horseshoe-shaped ring created by its six proteasome lid-CSN-initiation factor 3 (PCI) domain proteins, and a large bundle formed by the carboxy-terminal α-helices of every subunit. CSN5 and its dimerization partner, CSN6, are intricately embedded at the core of the helical bundle. In the substrate-free holoenzyme, CSN5 is autoinhibited, which precludes access to the active site. We find that neddylated CRL binding to CSN is sensed by CSN4, and communicated to CSN5 with the assistance of CSN6, resulting in activation of the deneddylase.

Structure of the DDB1-CRBN E3 ubiquitin ligase in complex with thalidomide.

In the 1950s, the drug thalidomide, administered as a sedative to pregnant women, led to the birth of thousands of children with multiple defects. Despite the teratogenicity of thalidomide and its derivatives lenalidomide and pomalidomide, these immunomodulatory drugs (IMiDs) recently emerged as effective treatments for multiple myeloma and 5q-deletion-associated dysplasia. IMiDs target the E3 ubiquitin ligase CUL4-RBX1-DDB1-CRBN (known as CRL4(CRBN)) and promote the ubiquitination of the IKAROS family transcription factors IKZF1 and IKZF3 by CRL4(CRBN). Here we present crystal structures of the DDB1-CRBN complex bound to thalidomide, lenalidomide and pomalidomide. The structure establishes that CRBN is a substrate receptor within CRL4(CRBN) and enantioselectively binds IMiDs. Using an unbiased screen, we identified the homeobox transcription factor MEIS2 as an endogenous substrate of CRL4(CRBN). Our studies suggest that IMiDs block endogenous substrates (MEIS2) from binding to CRL4(CRBN) while the ligase complex is recruiting IKZF1 or IKZF3 for degradation. This dual activity implies that small molecules can modulate an E3 ubiquitin ligase and thereby upregulate or downregulate the ubiquitination of proteins.

Searching for DNA lesions: structural evidence for lower- and higher-affinity DNA binding conformations of human alkyladenine DNA glycosylase.

To efficiently repair DNA, human alkyladenine DNA glycosylase (AAG) must search the million-fold excess of unmodified DNA bases to find a handful of DNA lesions. Such a search can be facilitated by the ability of glycosylases, like AAG, to interact with DNA using two affinities: a lower-affinity interaction in a searching process and a higher-affinity interaction for catalytic repair. Here, we present crystal structures of AAG trapped in two DNA-bound states. The lower-affinity depiction allows us to investigate, for the first time, the conformation of this protein in the absence of a tightly bound DNA adduct. We find that active site residues of AAG involved in binding lesion bases are in a disordered state. Furthermore, two loops that contribute significantly to the positive electrostatic surface of AAG are disordered. Additionally, a higher-affinity state of AAG captured here provides a fortuitous snapshot of how this enzyme interacts with a DNA adduct that resembles a one-base loop.

The molecular basis of CRL4DDB2/CSA ubiquitin ligase architecture, targeting, and activation.

The DDB1-CUL4-RBX1 (CRL4) ubiquitin ligase family regulates a diverse set of cellular pathways through dedicated substrate receptors (DCAFs). The DCAF DDB2 detects UV-induced pyrimidine dimers in the genome and facilitates nucleotide excision repair. We provide the molecular basis for DDB2 receptor-mediated cyclobutane pyrimidine dimer recognition in chromatin. The structures of the fully assembled DDB1-DDB2-CUL4A/B-RBX1 (CRL4(DDB2)) ligases reveal that the mobility of the ligase arm creates a defined ubiquitination zone around the damage, which precludes direct ligase activation by DNA lesions. Instead, the COP9 signalosome (CSN) mediates the CRL4(DDB2) inhibition in a CSN5 independent, nonenzymatic, fashion. In turn, CSN inhibition is relieved upon DNA damage binding to the DDB2 module within CSN-CRL4(DDB2). The Cockayne syndrome A DCAF complex crystal structure shows that CRL4(DCAF(WD40)) ligases share common architectural features. Our data support a general mechanism of ligase activation, which is induced by CSN displacement from CRL4(DCAF) on substrate binding to the DCAF.

Histone methylation by PRC2 is inhibited by active chromatin marks.

The Polycomb repressive complex 2 (PRC2) confers transcriptional repression through histone H3 lysine 27 trimethylation (H3K27me3). Here, we examined how PRC2 is modulated by histone modifications associated with transcriptionally active chromatin. We provide the molecular basis of histone H3 N terminus recognition by the PRC2 Nurf55-Su(z)12 submodule. Binding of H3 is lost if lysine 4 in H3 is trimethylated. We find that H3K4me3 inhibits PRC2 activity in an allosteric fashion assisted by the Su(z)12 C terminus. In addition to H3K4me3, PRC2 is inhibited by H3K36me2/3 (i.e., both H3K36me2 and H3K36me3). Direct PRC2 inhibition by H3K4me3 and H3K36me2/3 active marks is conserved in humans, mouse, and fly, rendering transcriptionally active chromatin refractory to PRC2 H3K27 trimethylation. While inhibition is present in plant PRC2, it can be modulated through exchange of the Su(z)12 subunit. Inhibition by active chromatin marks, coupled to stimulation by transcriptionally repressive H3K27me3, enables PRC2 to autonomously template repressive H3K27me3 without overwriting active chromatin domains.

Detecting UV-lesions in the genome: The modular CRL4 ubiquitin ligase does it best!

The DDB1-DDB2-CUL4-RBX1 complex serves as the primary detection device for UV-induced lesions in the genome. It simultaneously functions as a CUL4 type E3 ubiquitin ligase. We review the current understanding of this dual function ubiquitin ligase and damage detection complex. The DDB2 damage binding module is merely one of a large family of possible DDB1-CUL4 associated factors (DCAF), most of which are substrate receptors for other DDB1-CUL4 complexes. DDB2 and the Cockayne-syndrome A protein (CSA) function in nucleotide excision repair, whereas the remaining receptors operate in a wide range of other biological pathways. We will examine the modular architecture of DDB1-CUL4 in complex with DDB2, CSA and CDT2 focusing on shared architectural, targeting and regulatory principles.

Structural basis for the inhibition of human alkyladenine DNA glycosylase (AAG) by 3,N4-ethenocytosine-containing DNA.

Reactive oxygen and nitrogen species, generated by neutrophils and macrophages in chronically inflamed tissues, readily damage DNA, producing a variety of potentially genotoxic etheno base lesions; such inflammation-related DNA damage is now known to contribute to carcinogenesis. Although the human alkyladenine DNA glycosylase (AAG) can specifically bind DNA containing either 1,N(6)-ethenoadenine (εA) lesions or 3,N(4)-ethenocytosine (εC) lesions, it can only excise εA lesions. AAG binds very tightly to DNA containing εC lesions, forming an abortive protein-DNA complex; such binding not only shields εC from repair by other enzymes but also inhibits AAG from acting on other DNA lesions. To understand the structural basis for inhibition, we have characterized the binding of AAG to DNA containing εC lesions and have solved a crystal structure of AAG bound to a DNA duplex containing the εC lesion. This study provides the first structure of a DNA glycosylase in complex with an inhibitory base lesion that is induced endogenously and that is also induced upon exposure to environmental agents such as vinyl chloride. We identify the primary cause of inhibition as a failure to activate the nucleotide base as an efficient leaving group and demonstrate that the higher binding affinity of AAG for εC versus εA is achieved through formation of an additional hydrogen bond between Asn-169 in the active site pocket and the O(2) of εC. This structure provides the basis for the design of AAG inhibitors currently being sought as an adjuvant for cancer chemotherapy.

Frameshift mutagenesis and microsatellite instability induced by human alkyladenine DNA glycosylase.

Human alkyladenine DNA glycosylase (hAAG) excises alkylated purines, hypoxanthine, and etheno bases from DNA to form abasic (AP) sites. Surprisingly, elevated expression of hAAG increases spontaneous frameshift mutagenesis. By random mutagenesis of eight active site residues, we isolated hAAG-Y127I/H136L double mutant that induces even higher rates of frameshift mutation than does the wild-type hAAG; the Y127I mutation accounts for the majority of the hAAG-Y127I/H136L-induced mutator phenotype. The hAAG-Y127I/H136L and hAAG-Y127I mutants increased the rate of spontaneous frameshifts by up to 120-fold in S. cerevisiae and also induced high rates of microsatellite instability (MSI) in human cells. hAAG and its mutants bind DNA containing one and two base-pair loops with significant affinity, thus shielding them from mismatch repair; the strength of such binding correlates with their ability to induce the mutator phenotype. This study provides important insights into the mechanism of hAAG-induced genomic instability.

Mutational studies of Pa-AGOG DNA glycosylase from the hyperthermophilic crenarchaeon Pyrobaculum aerophilum.

In all organisms studied to date, 8-oxoguanine (GO), an important oxidation product of guanine, is removed by highly conserved GO DNA glycosylases. The hyperthermophilic crenarchaeon Pyrobaculum aerophilum encodes a GO DNA glycosylase, Pa-AGOG (Archaeal GO DNA glycosylase) which has become the founding member of a new family within the HhH-GPD superfamily of DNA glycosylases based on unique structural and functional characteristics. In this study, we made quantitative measurements of the DNA glycosylase activity of Pa-AGOG wild type and some engineered variants under single turnover conditions. The mutagenesis study includes residues Trp222 (W222A and W222F), Trp69 (W69F), Gln31 (Q31S) and Lys147 (K147Q) all of which are involved in GO recognition and Asp172 (D172N and D172Q) and Lys140 (K140Q) that are involved in catalysis. Pa-AGOG prefers GO/G mispairs for both base excision and base excision/beta-lyase activities. The mutagenesis studies show that base-stacking between GO and Trp222 is very important for recognition. The contact between Trp69 and the 8-oxo group was found to be dispensable, while that to N7 by Gln31 is indispensable for GO recognition. In contrast to human OGG1 the catalytic mutant, D172Q did not show detectable glycosylase activity. Pa-AGOG mutants K140Q, D172N and D172Q did bind GO containing single-stranded DNA more tightly than double-stranded DNA containing a GO/C base pair. Our studies confirm and extend the unique characteristics of Pa-AGOG, which distinguish it from other mesophilic and thermostable GO DNA glycosylases.

Recognition and processing of a new repertoire of DNA substrates by human 3-methyladenine DNA glycosylase (AAG).

The human 3-methyladenine DNA glycosylase (AAG) recognizes and excises a broad range of purines damaged by alkylation and oxidative damage, including 3-methyladenine, 7-methylguanine, hypoxanthine (Hx), and 1,N(6)-ethenoadenine (epsilonA). The crystal structures of AAG bound to epsilonA have provided insights into the structural basis for substrate recognition, base excision, and exclusion of normal purines and pyrimidines from its substrate recognition pocket. In this study, we explore the substrate specificity of full-length and truncated Delta80AAG on a library of oligonucleotides containing structurally diverse base modifications. Substrate binding and base excision kinetics of AAG with 13 damaged oligonucleotides were examined. We found that AAG bound to a wide variety of purine and pyrimidine lesions but excised only a few of them. Single-turnover excision kinetics showed that in addition to the well-known epsilonA and Hx substrates, 1-methylguanine (m1G) was also excised efficiently by AAG. Thus, along with epsilonA and ethanoadenine (EA), m1G is another substrate that is shared between AAG and the direct repair protein AlkB. In addition, we found that both the full-length and truncated AAG excised 1,N(2)-ethenoguanine (1,N(2)-epsilonG), albeit weakly, from duplex DNA. Uracil was excised from both single- and double-stranded DNA, but only by full-length AAG, indicating that the N-terminus of AAG may influence glycosylase activity for some substrates. Although AAG has been primarily shown to act on double-stranded DNA, AAG excised both epsilonA and Hx from single-stranded DNA, suggesting the possible significance of repair of these frequent lesions in single-stranded DNA transiently generated during replication and transcription.

Substrate specificity and sequence-dependent activity of the Saccharomyces cerevisiae 3-methyladenine DNA glycosylase (Mag).

DNA glycosylases initiate base excision repair by first binding, then excising aberrant DNA bases. Saccharomyces cerevisiae encodes a 3-methyladenine (3MeA) DNA glycosylase, Mag, that recognizes 3MeA and various other DNA lesions including 1,N6-ethenoadenine (epsilon A), hypoxanthine (Hx) and abasic (AP) sites. In the present study, we explore the relative substrate specificity of Mag for these lesions and in addition, show that Mag also recognizes cisplatin cross-linked adducts, but does not catalyze their excision. Through competition binding and activity studies, we show that in the context of a random DNA sequence Mag binds epsilon A and AP-sites the most tightly, followed by the cross-linked 1,2-d(ApG) cisplatin adduct. While epsilon A binding and excision by Mag was robust in this sequence context, binding and excision of Hx was extremely poor. We further studied the recognition of epsilon A and Hx by Mag, when these lesions are present at different positions within A:T and G:C tracts. Overall, epsilon A was slightly less well excised from each position within the A:T and G:C tracts compared to excision from the random sequence, whereas Hx excision was greatly increased in these sequence contexts (by up to 7-fold) compared to the random sequence. However, given most sequence contexts, Mag had a clear preference for epsilon A relative to Hx, except in the TTXTT (X=epsilon A or Hx) sequence context from which Mag removed both lesions with almost equal efficiency. We discuss how DNA sequence context affects base excision by various 3MeA DNA glycosylases.

A DNA glycosylase from Pyrobaculum aerophilum with an 8-oxoguanine binding mode and a noncanonical helix-hairpin-helix structure.

Studies of DNA base excision repair (BER) pathways in the hyperthermophilic crenarchaeon Pyrobaculum aerophilum identified an 8-oxoguanine-DNA glycosylase, Pa-AGOG (archaeal GO glycosylase), with distinct functional characteristics. Here, we describe its crystal structure and that of its complex with 8-oxoguanosine at 1.0 and 1.7 A resolution, respectively. Characteristic structural features are identified that confirm Pa-AGOG to be the founding member of a functional class within the helix-hairpin-helix (HhH) superfamily of DNA repair enzymes. Its hairpin structure differs substantially from that of other proteins containing an HhH motif, and we predict that it interacts with the DNA backbone in a distinct manner. Furthermore, the mode of 8-oxoguanine recognition, which involves several hydrogen-bonding and pi-stacking interactions, is unlike that observed in human OGG1, the prototypic 8-oxoguanine HhH-type DNA glycosylase. Despite these differences, the predicted kinked conformation of bound DNA and the catalytic mechanism are likely to resemble those of human OGG1.

Pa-AGOG, the founding member of a new family of archaeal 8-oxoguanine DNA-glycosylases.

Oxidative damage represents a major threat to genomic stability, as the major product of DNA oxidation, 8-oxoguanine (GO), frequently mispairs with adenine during replication. In order to prevent these mutagenic events, organisms have evolved GO-DNA glycosylases that remove this oxidized base from DNA. We were interested to find out how GO is processed in the hyperthermophilic archaeon Pyrobaculum aerophilum, which lives at temperatures around 100 degrees C. To this end, we searched its genome for open reading frames (ORFs) bearing the principal hallmark of GO-DNA glycosylases: a helix-hairpin-helix motif and a glycine/proline-rich sequence followed by an absolutely conserved aspartate (HhH-GPD motif). Interestingly, although the P.aerophilum genome encodes three such ORFs, none of these encodes the potent GO-processing activity detected in P.aerophilum extracts. Fractionation of the extracts, followed by analysis of the active fractions by denaturing polyacrylamide gel electrophoresis, showed that the GO-processing enzyme has a molecular size of approximately 30 kDa. Mass spectrometric analysis of proteins in this size range identified several peptides originating from P.aerophilum ORF PAE2237. We now show that PAE2237 encodes AGOG (Archaeal GO-Glycosylase), the founding member of a new family of DNA glycosylases, which can remove GO from single- and double-stranded substrates with great efficiency.