Landon Pastushok

Crystal structure of the human ubiquitin conjugating enzyme complex, hMms2–hUbc13

The ubiquitin conjugating enzyme complex Mms2–Ubc13 plays a key role in post-replicative DNA repair in yeast and the NF-κB signal transduction pathway in humans. This complex assembles novel polyubiquitin chains onto yet uncharacterized protein targets. Here we report the crystal structure of a complex between hMms2 (Uev1) and hUbc13 at 1.85 Å resolution and a structure of free hMms2 at 1.9 Å resolution. These structures reveal that the hMms2 monomer undergoes a localized conformational change upon interaction with hUbc13. The nature of the interface provides a physical basis for the preference of Mms2 for Ubc13 as a partner over a variety of other structurally similar ubiquitin-conjugating enzymes. The structure of the hMms2–hUbc13 complex provides the conceptual foundation for understanding the mechanism of Lys 63 multiubiquitin chain assembly and for its interactions with the RING finger proteins Rad5 and Traf6.

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 κB (NF-κB) activation, whereas Ubc13–Uev1A is involved in NF-κB 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.

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.

The TRAF6 RING finger domain mediates physical interaction with Ubc13

Tumor necrosis factor receptor associated factor 6 (TRAF6) is an important signaling molecule involved in a diverse array of physiological processes. It has been proposed that TRAF6, a RING finger‐containing protein, acts as a ubiquitin ligase (E3) and a target for Lys‐63 linked polyubiquitination mediated by Ubc13–Uev, a ubiquitin conjugating (E2) complex. However, the physical interaction between TRAF6 and this E2 complex has not been reported. We used the yeast two‐hybrid assay to demonstrate that TRAF6 indeed interacts with the E2 complex through its direct binding to Ubc13. Either a single Cys‐to‐Ser substitution within the TRAF6 RING finger domain or an amino acid substitution on the Ubc13 surface, that is predicted to interact with RING finger proteins, is able to abolish the interaction. In addition, we found that TRAF6 can interact with itself and this self‐interaction domain is mapped to the N‐terminus containing the RING finger motif. Based on this study and our previous Ubc13–Uev structural analysis, the interface of Ubc13‐TRAF6 RING finger can be predicted.

DNA damage-induced gene expression in Saccharomyces cerevisiae

After exposure to DNA-damaging agents, both prokaryotic and eukaryotic cells activate stress responses that result in specific alterations in patterns of gene expression. Bacteria such as Escherichia coli possess both lesion-specific responses as well as an SOS response to general DNA damage, and the molecular mechanisms of these responses are well studied. Mechanisms of DNA damage response in lower eukaryotes such as Saccharomyces cerevisiae are apparently different from those in bacteria. It becomes clear that many DNA damage-inducible genes are coregulated by the cell-cycle checkpoint, a signal transduction cascade that coordinates replication, repair, transcription and cell-cycle progression. On the other hand, among several well-characterized yeast DNA damage-inducible genes, their effectors and mechanisms of transcriptional regulation are rather different. This review attempts to summarize the current state of knowledge on the molecular mechanisms of DNA damage-induced transcriptional regulation in this model lower eukaryotic microorganism.

DNA postreplication repair modulated by ubiquitination and sumoylation.

Publisher Summary The living cell has developed ways to reduce or avoid detrimental changes to its genetic material. Numerous external and internal agents act and modify DNA. The incredible task of ensuring DNA fidelity and the survival of individual organisms is made possible by a variety of DNA repair and replication processes. It is conceivable that both prokaryotic and eukaryotic organisms employ a centrally controlled postreplicative survival mechanism to deal with ssDNA gaps left by replication blocks during DNA synthesis. The discovery of novel signal transduction mechanisms in postreplication repair (PRR) through the sequential modification of proliferating cell-nuclear antigen (PCNA) by two E2–E3 ubiquitination complexes sets an important milestone in the research of eukaryotic DNA repair. The Rad6–Rad18 complex bridges monoubiquitination of PCNA with error-prone postreplication repair (PRR), whereas Ubc13-Mms2-Rad5 ties novel Lys-63 polyubiquitination of PCNA with error-free PRR.

Arabidopsis thaliana Y-family DNA polymerase η catalyses translesion synthesis and interacts functionally with PCNA2

SUMMARY Upon blockage of chromosomal replication by DNA lesions, Y-family polymerases interact with monoubiquitylated proliferating cell nuclear antigen (PCNA) to catalyse translesion synthesis (TLS) and restore replication fork progression. Here, we assessed the roles of Arabidopsis thaliana POLH, which encodes a homologue of Y-family polymerase eta (Poleta), PCNA1 and PCNA2 in TLS-mediated UV resistance. A T-DNA insertion in POLH sensitized the growth of roots and whole plants to UV radiation, indicating that AtPoleta contributes to UV resistance. POLH alone did not complement the UV sensitivity conferred by deletion of yeast RAD30, which encodes Poleta, although AtPoleta exhibited cyclobutane dimer bypass activity in vitro, and interacted with yeast PCNA, as well as with Arabidopsis PCNA1 and PCNA2. Co-expression of POLH and PCNA2, but not PCNA1, restored normal UV resistance and mutation kinetics in the rad30 mutant. A single residue difference at site 201, which lies adjacent to the residue (lysine 164) ubiquitylated in PCNA, appeared responsible for the inability of PCNA1 to function with AtPoleta in UV-treated yeast. PCNA-interacting protein boxes and an ubiquitin-binding motif in AtPoleta were found to be required for the restoration of UV resistance in the rad30 mutant by POLH and PCNA2. These observations indicate that AtPoleta can catalyse TLS past UV-induced DNA damage, and links the biological activity of AtPoleta in UV-irradiated cells to PCNA2 and PCNA- and ubiquitin-binding motifs in AtPoleta.

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.

Constitutive fusion of ubiquitin to PCNA provides DNA damage tolerance independent of translesion polymerase activities

In response to replication-blocking DNA lesions, proliferating cell nuclear antigen (PCNA) can be conjugated with a single ubiquitin (Ub) or Lys63-linked Ub chains at the Lys164 residue, leading to two modes of DNA damage tolerance (DDT), namely translesion synthesis (TLS) and error-free DDT, respectively. Several reports suggest a model whereby monoubiquitylated PCNA recruits TLS polymerases through an enhanced physical association. We sought to examine this model in Saccharomyces cerevisiae through artificial fusions of Ub to PCNA in vivo. We created N- and C- terminal gene fusions of Ub to PCNA-K164R (collectively called PCNA·Ub) and found that both conferred tolerance to DNA damage. The creation of viable PCNA·Ub strains lacking endogenous PCNA enabled a thorough analysis of roles for PCNA mono-Ub in DDT. As expected, the DNA damage resistance provided by PCNA·Ub is not dependent on RAD18 or UBC13. Surprisingly, inactivation of TLS polymerases did not abolish PCNA·Ub resistance to DNA damage, nor did PCNA·Ub cause elevated spontaneous mutagenesis, which is a defining characteristic of REV3-dependent TLS activity. Taken together, our data suggest that either the monoubiquitylation of PCNA does not promote TLS activity in all cases or PCNA·Ub reveals a currently undiscovered role for monoubiquitylated PCNA in DNA damage tolerance.

Mechanisms of Activation of Phosphoenolpyruvate Carboxykinase from Escherichia coli by Ca2+ and of Desensitization by Trypsin

The 1.8-A resolution structure of the ATP-Mg(2+)-Ca(2+)-pyruvate quinary complex of Escherichia coli phosphoenolpyruvate carboxykinase (PCK) is isomorphous to the published complex ATP-Mg(2+)-Mn(2+)-pyruvate-PCK, except for the Ca(2+) and Mn(2+) binding sites. Ca(2+) was formerly implicated as a possible allosteric regulator of PCK, binding at the active site and at a surface activating site (Glu508 and Glu511). This report found that Ca(2+) bound only at the active site, indicating that there is likely no surface allosteric site. (45)Ca(2+) bound to PCK with a K(d) of 85 micro M and n of 0.92. Glu508Gln Glu511Gln mutant PCK had normal activation by Ca(2+). Separate roles of Mg(2+), which binds the nucleotide, and Ca(2+), which bridges the nucleotide and the anionic substrate, are implied, and the catalytic mechanism of PCK is better explained by studies of the Ca(2+)-bound structure. Partial trypsin digestion abolishes Ca(2+) activation (desensitizes PCK). N-terminal sequencing identified sensitive sites, i.e., Arg2 and Arg396. Arg2Ser, Arg396Ser, and Arg2Ser Arg396Ser (double mutant) PCKs altered the kinetics of desensitization. C-terminal residues 397 to 540 were removed by trypsin when wild-type PCK was completely desensitized. Phe409 and Phe413 interact with residues in the Ca(2+) binding site, probably stabilizing the C terminus. Phe409Ala, DeltaPhe409, Phe413Ala, Delta397-521 (deletion of residues 397 to 521), Arg396(TAA) (stop codon), and Asp269Glu (Ca(2+) site) mutations failed to desensitize PCK and, with the exception of Phe409Ala, appeared to have defects in the synthesis or assembly of PCK, suggesting that the structure of the C-terminal domain is important in these processes.