Arabidopsis UEV1D promotes Lysine-63-linked polyubiquitination and is involved in DNA damage response

DNA damage tolerance (DDT) in budding yeast requires Lys-63-linked polyubiquitination of the proliferating cell nuclear antigen. The ubiquitin-conjugating enzyme Ubc13 and the Ubc enzyme variant (Uev) methyl methanesulfonate2 (Mms2) are required for this process. Mms2 homologs have been found in all eukaryotic genomes examined; however, their roles in multicellular eukaryotes have not been elucidated. We report the isolation and characterization of four UEV1 genes from Arabidopsis thaliana. All four Uev1 proteins can form a stable complex with At Ubc13 or with Ubc13 from yeast or human and can promote Ubc13-mediated Lys-63 polyubiquitination. All four Uev1 proteins can replace yeast MMS2 DDT functions in vivo. Although these genes are ubiquitously expressed in most tissues, UEV1D appears to express at a much higher level in germinating seeds and in pollen. We obtained and characterized two uev1d null mutant T-DNA insertion lines. Compared with wild-type plants, seeds from uev1d null plants germinated poorly when treated with a DNA-damaging agent. Those that germinated grew slower, and the majority ceased growth within 2 weeks. Pollen from uev1d plants also displayed a moderate but significant decrease in germination in the presence of DNA damage. This report links Ubc13-Uev with functions in DNA damage response in Arabidopsis.     

Noncanonical MMS2-encoded ubiquitin-conjugating enzyme functions in assembly of novel polyubiquitin chains for DNA repair

Ubiquitin-conjugating enzyme variant (UEV) proteins resemble ubiquitin-conjugating enzymes (E2s) but lack the defining E2 active-site residue. The MMS2-encoded UEV protein has been genetically implicated in error-free postreplicative DNA repair in Saccharomyces cerevisiae. We show that Mms2p forms a specific heteromeric complex with the UBC13-encoded E2 and is required for the Ubc13p-dependent assembly of polyubiquitin chains linked through lysine 63. A ubc13 yeast strain is UV sensitive, and single, double, and triple mutants of the UBC13, MMS2, and ubiquitin (ubiK63R) genes display a comparable phenotype. These findings support a model in which an Mms2p/Ubc13p complex assembles novel polyubiquitin chains for signaling in DNA repair, and they suggest that UEV proteins may act to increase diversity and selectivity in ubiquitin conjugation.     

Drosophila Uev1a is dually required for Ben-dependent DNA-damage response and fly mobility

K63-linked polyubiquitination requires the ubiquitin-conjugating enzyme Ubc13 and a Ubc/E2 variant Uev. Lower eukaryotic organisms contain one UEV gene required for DNA-damage tolerance, while vertebrates and higher plants contain multiple UEV genes with distinct functions. In contrast, Drosophila contains only one UEV gene designated dUev1a. Here we report that dUev1a forms a stable heterodimer with Ben, the Drosophila Ubc13 ortholog, that dUev1a-F15E completely abolishes the interaction, and that a conserved dUev1a-F15Y substitution severely reduces its interaction with Ben. dUev1a functionally rescues the corresponding yeast mms2 null mutant from killing by various DNA-damaging agents in a Ben-dependent manner, and the heterozygous dUev1a mutant flies are more sensitive to DNA-damaging agent, indicating that the function of UEV in DNA-damage response is conserved throughout eukaryotes. Meanwhile, dUev1a^+/‐ mutant flies displayed reduced mobility characteristic of defects in the central nervous system and reminiscent of the bendless phenotypes, suggesting that dUev1a acts together with Ben in this process. Our observations collectively imply that dUev1a is dually required for DNA-damage response and neurological signaling in Drosophila, and that these processes are mediated by the Ben-dUev1a complex that promotes K63-linked polyubiquitination.     

Zebrafish Ubc13 is required for Lys63-linked polyubiquitination and DNA damage tolerance

Ubiquitination is an important post-translational protein modification that functions in diverse cellular processes of all eukaryotic organisms. Conventional Lys48-linked poly-ubiquitination leads to the degradation of specific proteins through 26S proteasomes, while Lys63-linked polyubiquitination appears to regulate protein activities in a non-proteolytic manner. To date, Ubc13 is the only known ubiquitin-conjugating enzyme capable of poly-ubiquitinating target proteins via Lys63-linked chains, and this activity absolutely requires a Ubc variant (Uev or Mms2) as a co-factor. However, Lys63-linked poly-ubiquitination and error-free DNA damage tolerance in zebrafish are yet to be defined. Here, we report molecular cloning and functional characterization of two zebrafish ubc13 genes, ubc13a and ubc13b. Analysis of their genomic structure, nucleotide and protein sequence indicates that the two genes are highly conserved during evolution and derived from whole genome duplication. Zebrafish Ubc13 proteins are able to physically interact with yeast or human Mms2 and both zebrafish ubc13 genes are able to functionally complement the yeast ubc13 null mutant for spontaneous mutagenesis and sensitivity to DNA damaging agents. In addition, upon DNA damage, the expression of zebrafish ubc13a and ubc13b is induced during embryogenesis and zebrafish Ubc13 is associated with nuclear chromatin. These results suggest the involvement of Lys63-linked poly-ubiquitylation in DNA damage response in zebrafish.     

Distinct regulation of Ubc13 functions by the two ubiquitin-conjugating enzyme variants Mms2 and Uev1A

Ubc13, a ubiquitin-conjugating enzyme (Ubc), requires the presence of a Ubc variant (Uev) for polyubiquitination. Uevs, although resembling Ubc in sequence and structure, lack the active site cysteine residue and are catalytically inactive. The yeast Uev (Mms2) incites noncanonical Lys63-linked polyubiquitination by Ubc13, whereas the increased diversity of Uevs in higher eukaryotes suggests an unexpected complication in ubiquitination. In this study, we demonstrate that divergent activities of mammalian Ubc13 rely on its pairing with either of two Uevs, Uev1A or Mms2. Structurally, we demonstrate that Mms2 and Uev1A differentially modulate the length of Ubc13-mediated Lys63-linked polyubiquitin chains. Functionally, we describe that Ubc13-Mms2 is required for DNA damage repair but not nuclear factor kappaB (NF-kappaB) activation, whereas Ubc13-Uev1A is involved in NF-kappaB activation but not DNA repair. Our finding suggests a novel regulatory mechanism in which different Uevs direct Ubcs to diverse cellular processes through physical interaction and alternative polyubiquitination.     

Ubc13 dosage is critical for immunoglobulin gene conversion and gene targeting in vertebrate cells

In contrast to lower eukaryotes, most vertebrate cells are characterized by a moderate efficiency of homologous recombination (HR) and limited feasibility of targeted genetic modifications. As a notable exception, the chicken DT40 B cell line is distinguished by efficient homology-mediated repair of DNA lesions during Ig gene conversion, and also shows exceptionally high gene-targeting efficiencies. The molecular basis of these phenomena is elusive. Here we show that the activity levels of Ubc13, the E2 enzyme responsible for non-canonical K63-linked polyubiquitination, are critical for high efficiency of Ig gene conversion and gene targeting in DT40. Ubc13^+/− cells show substantially lower homology-mediated repair, yet do not display changes in somatic hypermutation, overall DNA repair or cell proliferation. Our results suggest that modulation of the activity of K63-linked polyubiquitination may be used to customize HR efficiencies in vertebrate cells.     

Roles of mouse UBC13 in DNA postreplication repair and Lys63-linked ubiquitination

The E2 enzyme, Ubc13, and the E2 enzyme variants, Uevs, form stable, high affinity complexes for the assembly of Lys63-linked ubiquitin chains. This process is involved in error-free DNA postreplication repair, the activation of kinases in the NF-kappaB signaling pathway and possibly other cellular processes. To further investigate the roles played by Ubc13 in a whole animal model, we report here the molecular cloning of mouse UBC13 and show for the first time that a mammalian UBC13 gene is able to complement the yeast ubc13 null mutant. Furthermore, in vitro analyses and a yeast two-hybrid assay show that mUbc13 is able to form stable complexes with various Uevs. In the presence of E1 and ATP, mUbc13 forms thiolesters with ubiquitin; however, the formation of Lys63-linked di-ubiquitin and multi-ubiquitin chains is dependent on Uevs. These results suggest that the roles of UBC13 are conserved throughout eukaryotes and that the mouse is an appropriate model for the study of Ubc13-mediated Lys63-linked ubiquitin signaling pathways in humans.     

Mms2-Ubc13 covalently bound to ubiquitin reveals the structural basis of linkage-specific polyubiquitin chain formation

Lys63-linked polyubiquitin chains participate in nonproteolytic signaling pathways, including regulation of DNA damage tolerance and NF-kappaB activation. E2 enzymes bound to ubiquitin E2 variants (UEV) are vital in these pathways, synthesizing Lys63-linked polyubiquitin chains, but how these complexes achieve specificity for a particular lysine linkage has been unclear. We have determined the crystal structure of an Mms2-Ubc13-ubiquitin (UEV-E2-Ub) covalent intermediate with donor ubiquitin linked to the active site residue of Ubc13. In the structure, the unexpected binding of a donor ubiquitin of one Mms2-Ubc13-Ub complex to the acceptor-binding site of Mms2-Ubc13 in an adjacent complex allows us to visualize at atomic resolution the molecular determinants of acceptor-ubiquitin binding. The structure reveals the key role of Mms2 in allowing selective insertion of Lys63 into the Ubc13 active site and suggests a molecular model for polyubiquitin chain elongation.     

Structural Basis for Non-Covalent Interaction Between Ubiquitin and the Ubiquitin Conjugating Enzyme Variant Human MMS2

Modification of proteins by post-translational covalent attachment of a single, or chain, of ubiquitin molecules serves as a signaling mechanism for a number of regulatory functions in eukaryotic cells. For example, proteins tagged with lysine-63 linked polyubiquitin chains are involved in error-free DNA repair. The catalysis of lysine-63 linked polyubiquitin chains involves the sequential activity of three enzymes (E1, E2, and E3) that ultimately transfer a ubiquitin thiolester intermediate to a protein target. The E2 responsible for catalysis of lysine-63 linked polyubiquitination is a protein heterodimer consisting of a canonical E2 known as Ubc13, and an E2-like protein, or ubiquitin conjugating enzyme variant (UEV), known as Mms2. We have determined the solution structure of the complex formed by human Mms2 and ubiquitin using high resolution, solution state nuclear magnetic resonance (NMR) spectroscopy. The structure of the Mms2–Ub complex provides important insights into the molecular basis underlying the catalysis of lysine-63 linked polyubiquitin chains.     

Arabidopsis thaliana UBC13: Implication of Error-free DNA Damage Tolerance and Lys63-linked Polyubiquitylation in Plants

Ubiquitylation is an important biochemical reaction found in all eukaryotic organisms and is involved in a wide range of cellular processes. Conventional ubiquitylation requires the formation of polyubiquitin chains linked through Lys48 of the ubiquitin, which targets specific proteins for degradation. Recently polyubiquitylation through a noncanonical Lys63 chain has been reported, and is required for error-free DNA damage tolerance (or postreplication repair) in yeast. To date, Ubc13 is the only known ubiquitin-conjugating enzyme (Ubc) capable of catalyzing the Lys63-linked polyubiquitylation reaction and this function requires interaction with the Ubc variant Mms2. No information is available on either Lys63-linked ubiquitylation or error-free damage tolerance in plants. We thus cloned and functionally characterized two Arabidopsis thaliana UBC13 genes, AtUBC13A and AtUBC13B. The two genes are highly conserved with respect to chromosomal structure and protein sequence, suggesting that they are derived from a recent gene duplication event. Both AtUbc13 proteins are able to physically interact with yeast or human Mms2, implying that plants also employ the Lys63-linked polyubiquitylation reaction. Furthermore, AtUBC13 genes are able to functionally complement the yeast ubc13 null mutant for spontaneous mutagenesis and sensitivity to DNA damaging agents, suggesting the existence of an error-free DNA damage tolerance pathway in plants. The AtUBC13 genes appear to express ubiquitously and are not induced by various conditions tested.     

Ubiquitin binding site of the ubiquitin E2 variant (UEV) protein Mms2 is required for DNA damage tolerance in the yeast RAD6 pathway

Different ubiquitin modifications to proliferating cell nuclear antigen (PCNA) signal distinct modes of lesion bypass in the RAD6 pathway of DNA damage tolerance. The modification of PCNA with monoubiquitin signals an error-prone bypass, whereas the extension of this modification into a Lys-63-linked polyubiquitin chain promotes error-free bypass. Chain formation is catalyzed by the Mms2/Ubc13 conjugating enzyme variant/conjugating enzyme (UEV.E2) complex together with the Rad5 ubiquitin ligase. In vitro studies of this UEV.E2 complex have identified a ubiquitin binding site that is mainly localized on Mms2. However, the role of this site in DNA damage tolerance and the molecular features of the ubiquitin/Mms2 interaction are poorly understood. Here we identify two molecular determinants, the side chains of Mms2-Ile-57 and ubiquitin-Ile-44, that are required for chain assembly in vitro and error-free lesion bypass in vivo. Mutating either of these side chains to alanine elicits a severe 10-20-fold inhibition of chain synthesis that is caused by compromised binding of the acceptor ubiquitin to Mms2. These results suggest that the ubiquitin binding site of Mms2 is necessary for error-free lesion bypass in the RAD6 pathway and provide new insights into ubiquitin recognition by UEV proteins.     

Activation of TAK1 by Sef-S induces apoptosis in 293T cells

Sef (similar expression to fgf genes, also named IL-17RD) was identified as a negative regulator of fibroblast growth factor signaling. Sef-S, an alternative splice isoform of Sef, inhibits FGF-induced NIH3T3 cell proliferation. Here we report that Sef-S physically interacts with TAK1, induces Lys63-linked TAK1 polyubiquitination on lysine 209 and TAK1-mediated JNK and p38 activation. Co-overexpression of TAK1 WT, K34R, K150R, K158R mutants with Sef-S induces Lys63-linked TAK1 polyubiquitination whereas TAK1 K63R and K209R mutants fail. Furthermore, co-overexpression of Sef-S and TAK1 induce 293T cells apoptosis. These results reveal Sef-S actives Lys63-linked TAK1 polyubiquitination on lysine 209, induces TAK1-mediated JNK and p38 activation and also results apoptosis in 293T cells.     

A single Mms2 "key" residue insertion into a Ubc13 pocket determines the interface specificity of a human Lys63 ubiquitin conjugation complex

Human Ubc13 and Mms2 (or its homolog, Uev1) form a unique ubiquitin-conjugating enzyme (Ubc) complex that generates atypical Lys(63)-linked ubiquitin conjugates. Such conjugates are attached to specific targets that modulate the activity of various cellular processes including DNA repair, mitotic progression, and nuclear factor-kappaB signaling. Whereas Ubc13 is a typical Ubc, Mms2 is a non-catalytic Ubc variant. Substantial biochemical evidence has revealed a mechanism whereby Mms2 properly orients ubiquitin to allow for Lys(63) conjugation by Ubc13; however, how this specific Ubc13-Mms2 complex is formed and why Mms2 does not form a complex with other Ubcs have not been reported. In order to address these questions, we used a structure-based approach to design mutations and characterize the human Ubc13-Mms2 interface. We used the yeast two-hybrid assay, glutathione S-transferase pull-downs, and surface plasmon resonance to test in vivo and in vitro binding. These experiments were paired with functional complementation and ubiquitin conjugation studies to provide in vivo and in vitro functional data. The results in this study allowed us to identify important residues of the Ubc13-Mms2 interface, determine a correlation between heterodimer formation and function, and conclude why Mms2 forms a specific complex with Ubc13 but not other Ubc proteins.     

Tumor necrosis factor (TNF)-induced germinal center kinase-related (GCKR) and stress-activated protein kinase (SAPK) activation depends upon the E2/E3 complex Ubc13-Uev1A/TNF receptor-associated factor 2 (TRAF2)

Tumor necrosis factor (TNF)-induced activation of apoptosis signal-regulating kinase 1 (ASK1) and germinal center kinases (GCKs) and the subsequent activation of stress-activated protein kinases (SAPKs and c-Jun NH(2)-terminal kinases) requires TNF receptor-associated factor 2 (TRAF2). Although the TRAF2 TRAF domain binds ASK1, GCK, and the highly related kinase GCKR, the RING finger domain is needed for their activation. Here, we report that TNF activates GCKR and the SAPK pathway in a manner that depends upon TRAF2 and Ubc13, a member along with Uev1A of a dimeric ubiquitin-conjugating enzyme complex. Interference with Ubc13 function or expression inhibits both TNF- and TRAF2-mediated GCKR and SAPK activation, but has a minimal effect on ASK1 activation. TNF signaling leads to TRAF2 polyubiquitination and oligomerization and to the oligomerization, ubiquitination, and activation of GCKR, all of which are sensitive to the disruption of Ubc13 function. These results indicate that the assembly of a TRAF2 lysine 63-linked polyubiquitin chain by Ubc13/Uev1A is required for TNF-mediated GCKR and SAPK activation, but may not be required for ASK1 activation.     

The essential Ubc4/Ubc5 function in yeast is HECT E3-dependent, and RING E3-dependent pathways require only monoubiquitin transfer by Ubc4

The ubiquitin (Ub)-conjugating enzymes Ubc4 and Ubc5 are involved in a variety of ubiquitination pathways in yeast, including Rsp5- and anaphase-promoting complex (APC)-mediated pathways. We have found the double deletion of UBC4 and UBC5 genes in yeast to be lethal. To investigate the essential pathway disrupted by the ubc4/ubc5 deletion, several point mutations were inserted in Ubc4. The Ubc4 active site mutation C86A and the E3-binding mutations A97D and F63A were both unable to rescue the lethal phenotype, indicating that an active E3/E2～Ub complex is required for the essential function of Ubc4/Ubc5. A mutation that specifically eliminates RING E3-catalyzed isopeptide formation but not HECT E3 transthiolation (N78S-Ubc4) rescued the lethal phenotype. Thus, the essential redundant function performed by Ubc4 and Ubc5 in yeast is with a HECT-type E3, likely the only essential HECT in yeast, Rsp5. Our results also suggest that Ubc1 can weakly replace Ubc4 to transfer mono-Ub with APC, but Ubc4 cannot replace Ubc1 for poly-Ub chain extension on APC substrates. Finally, the backside Ub-binding mutant S23R-Ubc4 has no observable effect in yeast. Together, our results are consistent with a model in which Ubc4 and Ubc5 are 1) the primary E2s for Rsp5 in yeast and 2) act as monoubiquitinating E2s in RING E3-catalyzed pathways, in contrast to the processive human ortholog UbcH5.     

The Yeast Shu Complex Utilizes Homologous Recombination Machinery for Error-free Lesion Bypass via Physical Interaction with a Rad51 Paralogue

DNA-damage tolerance (DDT) is defined as a mechanism by which eukaryotic cells resume DNA synthesis to fill the single-stranded DNA gaps left by replication-blocking lesions. Eukaryotic cells employ two different means of DDT, namely translesion DNA synthesis (TLS) and template switching, both of which are coordinately regulated through sequential ubiquitination of PCNA at the K164 residue. In the budding yeast Saccharomyces cerevisiae, the same PCNA-K164 residue can also be sumoylated, which recruits the Srs2 helicase to prevent undesired homologous recombination (HR). While the mediation of TLS by PCNA monoubiquitination has been extensively characterized, the method by which K63-linked PCNA polyubiquitination leads to template switching remains unclear. We recently identified a yeast heterotetrameric Shu complex that couples error-free DDT to HR as a critical step of template switching. Here we report that the Csm2 subunit of Shu physically interacts with Rad55, an accessory protein involved in HR. Rad55 and Rad57 are Rad51 paralogues and form a heterodimer to promote Rad51-ssDNA filament formation by antagonizing Srs2 activity. Although Rad55-Rad57 and Shu function in the same pathway and both act to inhibit Srs2 activity, Shu appears to be dedicated to error-free DDT while the Rad55-Rad57 complex is also involved in double-strand break repair. This study reveals the detailed steps of error-free lesion bypass and also brings to light an intrinsic interplay between error-free DDT and Srs2-mediated inhibition of HR.     

TAK1 lysine 158 is required for TGF-β-induced TRAF6-mediated Smad-independent IKK/NF-κB and JNK/AP-1 activation

Lys63-linked TAK1 polyubiquitination plays an essential role in the regulation of TAK1 activation. TRAF6-mediated Lys63-linked polyubiquitylation of TAK1 has been shown to be required for TGF-β-induced TAK1 activation. However, it remains unclear which lysine residue on TAK1 is TRAF6-mediated TAK1 polyubiquitination acceptor site in TGF-β signaling pathway. Here we report that lysine 158 on TAK1 is required for TGF-β-induced TRAF6-mediated TAK1 polyubiquitination and TAK1-mediated IKK, JNK and p38 activation. Notably, in contrast to TAK1 wild-type and K34R mutant, TAK1 K158R mutant co-overexpression with TAB1 failed to induce Lys63-linked TAK1 polyubiquitination. TRAF6-induced K63-linked TAK1 polyubiquitination was blocked by TAK1 K158R mutation, but not by K34R mutation. Furthermore, TGF-β-induced TAK1 polyubiquitination was inhibited by TAK1 K158R mutation, but not by K34R mutation in HeLa cells. Reconstitution of TAK1-deficient mouse embryo fibroblast cells with TAK1 wild-type, K158R mutant, or K34R mutant reveals that TAK1 lysine 158 residue is required for TGF-β-induced IKK, p38 and JNK activation.     

Tumor suppressor ARF promotes non-classic proteasome-independent polyubiquitination of COMMD1

Although the tumor suppressor ARF is generally accepted for its essential role in activating the p53 pathway, its p53-independent function has also been proposed. Here, we report that ARF associates with COMMD1 and promotes Lys(63)-mediated polyubiquitination of COMMD1 in a p53-independent manner. We found that ARF interacts with COMMD1 in vivo. Deletion analysis of ARF suggested that the N-terminal amino acids 15-45 are important for its interaction with COMMD1. In addition, we found that endogenous ARF redistributes from the nucleolus to the nucleoplasm and interacts with COMMD1 when DNA is damaged by actinomycin D. Interestingly, we found that ARF promotes the polyubiquitination of COMMD1 through Lys(63) of ubiquitin but not the polyubiquitination of Lys(48), which does not target COMMD1 for proteasome-dependent proteolysis. Moreover, ARF mutants lacking the domain interacting with COMMD1 did not promote COMMD1 polyubiquitination, indicating that physical association is a prerequisite condition for the polyubiquitination process. Together, these data suggest that the ability to promote Lys(63)-mediated polyubiquitination of COMMD1 is a novel property of ARF independent of p53.