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First published online January 4, 2008; 10.1105/tpc.107.051862 The Plant Cell 20:213-227 (2008) © 2008 American Society of Plant Biologists Arabidopsis UEV1D Promotes Lysine-63–Linked Polyubiquitination and Is Involved in DNA Damage Response[W]
a Department of Microbiology and Immunology, University of Saskatchewan, Saskatoon, Saskatchewan, Canada S7N 5E5 3 Address correspondence to hong.wang{at}usask.ca or wei.xiao{at}usask.ca.
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.
Cellular DNA is subject to assaults by environmental factors and endogenous metabolites. The alteration of DNA can lead to mutagenesis, genome rearrangements, and cell death (Friedberg et al., 2006  ). To maintain genome integrity, all living organisms have evolved a variety of DNA repair mechanisms to protect cells from DNA damage. However, despite great advances made during the last decade in the field of DNA repair and mutagenesis, the molecular mechanisms of DNA damage tolerance (DDT) in eukaryotes, especially in multicellular eukaryotes, have not yet been well characterized. In the lower eukaryote Saccharomyces cerevisiae, a DDT process known as DNA postreplication repair (PRR) facilitates DNA synthesis in the presence of replication-blocking lesions in the template. PRR consists of two branches: an error-prone (mutagenesis) branch and an error-free branch. The error-prone branch is mediated by specialized DNA polymerases, including Pol (Rev3 + Rev7), Pol , and Rev1, which are required for translesion DNA synthesis (TLS). By contrast, the error-free branch is mediated by the ubiquitin (Ub)-conjugating enzyme (Ubc or E2)–Ubc variant (Uev) complex Ubc13-Mms2 (for methyl methanesulfonate2), which acts to prevent spontaneous and DNA damage–induced mutagenesis (Broomfield et al., 2001; Barbour and Xiao, 2003). It is now clear that yeast PRR is accomplished by stepwise covalent modifications of the proliferating cell nuclear antigen (PCNA) encoded by POL30. In response to DNA damage, the Rad6-Rad18 ubiquitination complex monoubiquitinates Pol30 at the Lys-164 residue, then the Mms2-Ubc13-Rad5 complex further polyubiquitinates Pol30 through Lys-63–linked chains (Hoege et al., 2002). It is thus assumed that monoubiquitinated Pol30 promotes error-prone TLS, whereas polyubiquitinated Pol30 promotes error-free bypass of damaged templates (Stelter and Ulrich, 2003; Pastushok and Xiao, 2004). The same Lys-164 residue can also be covalently modified by SUMO (for small ubiquitin-related modifier), which requires the Siz1-Ubc9 complex; sumoylated Pol30 recruits the DNA helicase Srs2 to stalled replication forks to prevent inappropriate recombination (Papouli et al., 2005; Pfander et al., 2005).
Ubc13 and Uev homologs are found in all eukaryotes examined to date (Villalobo et al., 2002
Protein ubiquitination and its role in regulating protein degradation have been extensively studied in plants (Bachmair et al., 2001
Arabidopsis genes involved in TLS, including REV3 (Sakamoto et al., 2003
Isolation of Arabidopsis UEV1 Genes To identify Arabidopsis UEV1 genes, a human Mms2 sequence (Xiao et al., 1998 ) was used to search for homologs in the Arabidopsis protein database (through The Arabidopsis Information Resource [TAIR]; www.arabidopsis.org). Four hypothetical proteins with a high degree of similarity (E-values < 2e-38) were found and named At Uev1A (At1g23260), At Uev1B (At1g70660), At Uev1C (At2g36060), and At Uev1D (At3g52560).
The genomic structures of these four corresponding UEV1 genes are shown in Figure 1A
. UEV1A and UEV1B have the same number of exons and introns, and their exon–intron junctions are identical. UEV1B has shorter intron sequences than UEV1A. Similarly, UEV1C and UEV1D have the same number of exons and introns, the same exon–intron junctions, and all introns in UEV1D are longer than those in UEV1C. Nucleotide sequence alignment of UEV1 open reading frames (ORFs) reveals 86% identity between UEV1A and UEV1B and 88% identity between UEV1C and UEV1D. Based on the above analyses, we predict that the four UEV1 genes resulted from two separate gene duplication events. This agrees with a duplication mapping analysis (http://wolfe.gen.tcd.ie/athal/dup) (Blanc et al., 2003
All four UEV1 ORFs were cloned from Arabidopsis by RT-PCR using gene-specific primers. The nucleotide sequences were identical to the annotated complete coding sequences in the Arabidopsis database. The predicted Uev1A, -B, -C, and -D proteins contain 158, 159, 145, and 146 amino acids, respectively, with differences in length primarily at the N or C terminus. Uev1A and Uev1B contain C-terminal tails not found in other Uevs (Figure 1B). Amino acid sequence alignment (Figure 1B) shows 86% identity between Uev1A and Uev1B and 92% identity between Uev1C and AtUev1D, whereas amino acid sequence identity between the two pairs is  75%.
The sequences of At Uev1 proteins were also aligned with those of Uev proteins from six other eukaryotic organisms, including human. As shown in Figure 1B, amino acid sequence identity between At Uev1s and those from other species ranges from 47 to 56%, and similarity ranges from 65 to 75%. Furthermore, several critical residues implicated in Uev activity are also conserved in At Uev1s. These residues include Phe-13 of Hs Mms2 required for physical interaction with Ubc13 (Pastushok et al., 2005 Phylogenetic analysis (see Supplemental Figure 1 online) was performed on At Uev1s in relation to Uevs from the above model organisms as well as with other plant species of known genomic sequence. This analysis revealed that plant UEV1 genes evolved from a common UEV1/MMS2 ancestor, which were duplicated and further evolved within each species. Hence, it would be of great interest to examine whether or how functions have evolved in the UEV family of genes.
Physical Interaction of At Uev1 with Ubc13 from Different Species
To further confirm the physical interaction between Uev1 and Ubc13 in vitro, a glutathione S-transferase (GST)–affinity pull-down assay was conducted. As shown in Figure 2C, purified GST-Uev1A (lane 6) and GST-Uev1D (lane 7) are able to specifically interact with Ubc13A. As a negative control, GST alone (lane 5) does not bind to Ubc13A under the same experimental conditions. Similar results were also obtained with Uev1B and Uev1C (see Supplemental Figure 2B online). Hence, all four Uev1 proteins are able to form stable heterodimers with Ubc13.
Uev1 Is Required for Ubc13-Mediated Lys-63–Linked Polyubiquitination in Vitro
At UEV1 Genes Functionally Complement Yeast mms2 Null Mutants
The complementation of yeast mms2 relies on heterodimer formation between At Uev1 and yeast Ubc13. In order to assess in vivo complex formation and functions between Arabidopsis Uev1 and Ubc13, we created a yeast mms2 ubc13 double mutant and cotransformed it with At UEV1 and At UBC13. When the double mutant cells were transformed with only At UBC13 or At UEV1, the transformed cells did not display enhanced resistance to MMS (Figure 3B), indicating that both Ubc13 and a Uev are required for the DDT function. Interestingly, when combined with At UBC13, UEV1C and UEV1D completely restored the MMS resistance to the wild-type level, whereas UEV1A and UEV1B barely rescued the host cells (Figure 3B; see Supplemental Figure 2D online). This result is in sharp contrast with the observations that all At UEV1s functioned equally well in the complementation of yeast mms2 single mutant (Figure 3A) and that Uev1A/B displayed higher binding capacity than Uev1C/D in yeast two-hybrid assays (Figures 2A and 2B; see Supplemental Figure 2A online).
One of the most astonishing phenotypes of a yeast mms2 (Broomfield et al., 1998
At UEV1 Expression in Different Tissues and under Stresses Since UEV1 is presumed to be involved in DDT and the ubiquitination process is often involved in stress responses, we analyzed UEV1 expression under various stress conditions. Arabidopsis cell suspension culture was subjected to treatments as indicated and total RNA was isolated for RNA gel blot hybridization. The results from samples of 24-h treatments are presented in Supplemental Figure 3A online. It appears that UEV1 expression is slightly decreased after treatment with MMS or H2O2 and slightly increased after treatment with abscisic acid or mannitol, although for the latter two treatments the transcript level of the control UBQ11 was also higher.
Since all four UEV1 genes share >72% nucleotide sequence identity in their core coding region and all four predicted transcripts are similar in size, we suspect that the UEV1C probe used for RNA gel blot hybridization actually detected all four UEV1 transcripts. Given the fact that the two human Uev homologs (UEV1A and hMMS2) play distinct roles in cellular metabolism (Andersen et al., 2005 The expression of UEV1 genes in different tissues was also determined by RNA gel blot hybridization (see Supplemental Figure 3C online) and by analyzing the microarray data (Figure 4A ; see Supplemental Figure 4 online). While most tissues express variable levels of each UEV1 transcript, UEV1D appears to show a higher level of expression than the other three UEV1 genes in most tissues examined (Figure 4A; see Supplemental Figure 4 online). Greater differences in transcript levels of the UEV1 genes were found in samples from pollen and geminating seeds. The microarray data indicate that 3 h after seed germination, the expression of UEV1C and UEV1D is much higher than that of UEV1A and UEV1B and that UEV1D is essentially the only UEV1 transcript detected from pollen. To validate the microarray data, we performed RT-PCR with various tissues, including germinating seeds and pollen. Under the conditions used, the amount of PCR product was not excessive and was deemed to reflect the amount of cDNA template. Representative results are shown in Figure 4B and summarized as follows. First, all four UEV1 genes are indeed expressed in most common tissues, such as root, shoot, leaf, and stem. Second, only UEV1D transcript is detectable in pollen under our experimental conditions, consistent with the microarray data. Third, 6 h after seed germination, all transcripts except UEV1B are detected, while after 2 d of seed germination, only UEV1A and UEV1D transcripts are found, with UEV1D at a clearly higher level than UEV1A. Microarray data show little expression of UEV1A in 3-h germinating seeds, but we consistently observed UEV1A transcript by PT-PCR in the sample we used. These differences may be due to the conditions used in the microarray experiments and in this study.
uev1d Mutant Plants Are Sensitive to the DNA-Damaging Agent MMS The analysis of UEV1 expression as well as the observation that in combination with UBC13, UEV1D but not UEV1A could completely rescue the yeast ubc13 mms2 double mutant, prompted us to focus our attention on UEV1D. We reasoned that uev1d mutant plants may display compromised tolerance to DNA damage in pollen and during seed germination. The UEV1D T-DNA insertion line SALK_064912 was obtained from the ABRC (www.arabidopsis.org), and the allele was named uev1d-1. Sequence analysis revealed that the T-DNA was inserted in the first intron of UEV1D, with the left border oriented toward the 3' end of the gene (Figure 5A ). The gene-specific primers (SP1 and SP2) and a primer specific to the left border sequence (LB1) were used to confirm the insertion of T-DNA (Figure 5B). To further confirm that UEV1D expression was abolished by this T-DNA insertion, total RNA was extracted from seedlings of wild-type and homozygous uev1d-1 plants and analyzed by RT-PCR for the expression of four UEV1 genes. As shown in Figure 5D, a fragment corresponding to the UEV1D ORF could be amplified from wild-type plants but not from the uev1d-1 line, while the expression of the other three At UEV1 genes remained unaltered.
The homozygous uev1d-1 plants do not display apparent morphological variations. In order to investigate the possible role of UEV1D in protecting cells from DNA damage, we analyzed the effect of MMS on seed germination, considering that UEV1D is strongly expressed during seed germination. We examined three controls—wild-type Arabidopsis ecotype Columbia, a T-DNA insertion line (SALK_042050) not affecting UEV1 genes, and a wild type segregant line derived from the initial SALK_064912 seeds (1d-1WT)—along with the homozygous uev1d-1 T-DNA insertion line. Three parameters related to seed germination were surveyed. First, the percentage of seeds that germinated in the presence of various concentrations of MMS was scored after a 5-d incubation. Seeds from uev1d-1 plants were much more sensitive to MMS treatment than any of the three control plants, and this response was dose-dependent (Figure 6A ). By contrast, in the absence of MMS, the uev1d-1 seeds did not show a noticeable difference from controls in the percentage of seed germination. Second, it was observed that the homozygous uev1d-1 seedlings were dying relatively quickly in the presence of MMS and displayed bleached pale cotyledons rather than the normal green cotyledons. Thus, the percentage of germinated seeds with green cotyledons was scored after 13 d. The data clearly indicate that the uev1d-1 line had reduced numbers of viable seedlings in the presence of MMS. In particular, in the presence of 0.01% MMS, 75 to 90% of control seedlings were viable, as judged by green seedlings, compared with <15% viable uev1d-1 seedlings under the same growth condition (Figures 6B and 6C). Finally, the average fresh weight of 13-d uev1d-1 mutant seedlings was reduced compared with that in control seedlings after MMS treatments. More specifically, with 0.005% MMS treatment, even though almost all uev1d-1 seedlings remained green, they only had half the fresh weight of the wild-type seedlings (Figure 6D).
To ensure that the above observations were specific to the T-DNA insertion at UEV1D, we obtained the second UEV1D T-DNA insertion line SALK_052144 from the ABRC, in which the T-DNA was inserted in the third exon of UEV1D (Figure 5A). We confirmed the T-DNA insertion by genomic PCR (Figure 5C) and named it uev1d-2. RT-PCR analysis demonstrated that the UEV1D mRNA is absent in the homozygous uev1d-2 line (Figure 5D). Phenotypic analyses showed that, like uev1d-1, the uev1d-2 mutant is hypersensitive to MMS treatment during seed germination (Figures 6E and 6F). From these results, we conclude that UEV1D is required for tolerance to DNA damage during seed germination. To assess whether the observed uev1d mutant phenotype is specific to DNA damage or is triggered by general stress, we measured seed germination in the presence of up to 0.2 M NaCl. There was no significant difference in the response of wild-type and uev1d plants to the salt stress (data not shown). We also attempted to assess the role of UEV1D in pollen germination by measuring the percentage of pollen germination in the presence of MMS. As shown in Figure 7 , inactivation of UEV1D resulted in a moderate but significant decrease in pollen germination. In the presence of 0.005% MMS, 33% of wild-type pollens germinated, while only 20% of uev1d-1 pollens germinated after 8 h of incubation, indicating that UEV1D also plays a critical role in protecting pollen from environmental DNA damage.
uev1a Mutant Plants Do Not Display MMS Sensitivity Since UEV1A is the only other UEV1 gene expressed during seed germination, we were interested in the phenotypes of this mutant plant. Unfortunately, a uev1a T-DNA insertion mutant line is not available from the ABRC; instead, we found a line (FLAG_128G02) with a T-DNA insertion at the fourth exon (see Supplemental Figure 5A online) from the Institut Jean-Pierre Bourgin collection. We obtained this line, screened the segregants, and confirmed the homozygous uev1a mutant (uev1a-1) by both genomic PCR (see Supplemental Figure 5B online) and RT-PCR (see Supplemental Figure 5C online). Seed germination assays were performed under the same experimental conditions described above. uev1a-1 mutant plants did not display enhanced MMS sensitivity compared with its wild-type segregants or with the parental strain Ws-4 (data not shown). This result indicates that inactivation of UEV1A does not alter DDT during Arabidopsis seed germination.
Lys-63–linked polyubiquitination of target proteins is considered to be a fundamentally different process from conventional Lys-48–linked polyubiquitination, which targets proteins for degradation via the 26S proteasome (Hochstrasser, 1996 ; Pickart, 2001b). Instead, it is deemed analogous to other posttranslational regulatory processes, such as phosphorylation and sumoylation, that alter target protein activities. Although Lys-63–linked polyubiquitination has been reported to be involved in several cellular processes, including stress response (Arnason and Ellison, 1994), mitochondrial inheritance (Fisk and Yaffe, 1999), plasma membrane protein endocytosis (Galan and Haguenauer-Tsapis, 1997), ribosome function (Spence et al., 2000), innate immunity (Deng et al., 2000; Wang et al., 2001; Zhou et al., 2004), mitotic cell cycle checkpoint (Bothos et al., 2003), and DNA repair (Hofmann and Pickart, 1999; Hoege et al., 2002), to date Ubc13 is the only known E2 capable of mediating Lys-63–linked Ub chain assembly. Interestingly, genetic analyses to date define only three functions of Ubc13, namely PRR in yeast (Hoege et al., 2002) and possibly mammals (Andersen et al., 2005) through covalent modification of PCNA, activation of NF- B by polyubiquitinating NEMO/IKK in mammals (Zhou et al., 2004), and involvement in synaptic connectivity between the giant fiber and a motor neuron in Drosophila. Drosophila ubc13 is also known as bendless (Muralidhar and Thomas, 1993; Oh et al., 1994), although the molecular mechanism of Ubc13 activity in this case remains unclear. The unique feature of Ubc13 compared with other Ubcs is its ability to form a stable complex with a Uev, which is homologous to other Ubcs but lacks the active Cys residue (Broomfield et al., 1998; Sancho et al., 1998). This family of Uevs engages a noncovalent interaction with Ub (McKenna et al., 2001) and orients this acceptor Ub to allow its Lys-63 residue to be exposed to the C terminus of donor Ub covalently bound to Ubc13 (McKenna et al., 2003). It appears that in mammals, Uevs not only facilitate polyubiquitination but also serve as a regulatory subunit to promote the ubiquitination of different targets (Andersen et al., 2005). In this study, we identified four Arabidopsis genes that meet the criteria of encoding a Uev and found by sequence analysis that they are two pairs of duplicated genes. We argue that these UEV1 genes are paralogs and that they evolved from a common ancestor within plants, since sequence alignment could not distinguish which Uev1 pair is more related to one of the two mammalian Uevs (Uev1A or Mms2). This observation raises two major questions. First, do Ubc13-Uev complexes from different organisms confer a conserved function? Second, if gene duplications occurred after animal–plant separation, how are their functions preserved? In this study, we demonstrated that all At Uev1 proteins are able to form stable complexes with At Ubc13 and to promote Ubc13-mediated Lys-63–linked polyubiquitination. The only other known Arabidopsis Uev found to date is Cop10, which was identified as a negative regulator of photomorphogenesis and functions by promoting target protein degradation (Suzuki et al., 2002).
The observation that At UBC13 (Wen et al., 2006
Despite its predominant expression among the four UEV1 genes, inactivation of UEV1D caused only a very moderate compromise in pollen tube growth in the presence of a DNA-damaging agent. This probably reflects the lack of cell division during pollen germination, whereas DDT is expected to operate only on replicated DNA (Barbour and Xiao, 2003
In our opinion, this study provides an important step toward understanding Ubc13-Uev–mediated Lys-63 polyubiquitination in general and the mechanisms of DDT in particular in plants. Several questions remain to be addressed. First, is UEV1D the only UEV1 gene involved in error-free DDT? Second, is UEV1D also involved in other cellular processes? Third, what are the other cellular processes that also require Ubc13-Uev–mediated polyubiquitination? We feel that given the near identity in amino acid sequence and similar complementation phenotypes in yeast, Uev1C is likely involved in the same cellular processes as Uev1D. This could explain our failure to detect a DNA repair/tolerance defect in uev1d plants in a root growth assay (data not shown). Indeed, the phenotypes of uev1d mutant plants may be considered to be moderate, which is due to either the backup or residual expression of other UEV1 genes or the nature of the error-free DDT defect in plants. One important aspect of future work will be to identify a uev1c null mutant, combine this mutation with uev1d, examine various tissues for a DDT defect, and relate the results to the UEV1 expression profile. It is also interesting that the yeast mms2/ubc13 or rev1/rev3/rev7 single mutants are moderately sensitive to killing by DNA-damaging agents, but the combination of any two mutations from different pathways results in strong synergistic interactions (Broomfield et al., 1998
The two pairs of UEV1s may be involved in different functions in plants. Although it remains possible that Uev1A and Uev1B are involved in DDT as well, our observations favor the suggestion that the Uev1A/B pair is probably involved in other cellular processes unrelated to DNA damage response. First, although UEV1A is expressed during seed germination, inactivation of this gene does not result in compromised seed germination in the presence of MMS, in sharp contrast with the uev1d mutant lines. Second, in the presence of UBC13, the DDT activities of UEV1A/B are much lower than those of UEV1C/D in yeast cells, despite the fact that Uev1A/B interact with At Ubc13 and Ubc13 from other species very well and are able to fully complement the yeast mms2 mutant when yeast Ubc13 is present. This result also effectively rules out the possibility that partial complementation by UEV1A/B was due to their poor expression in yeast cells. Third, it is interesting that human Uev1A contains an additional N-terminal 25 amino acid residues and plays a distinct role from that of hMms2; it may be reverted to play a role in DDT when its N-terminal sequence is experimentally deleted (Andersen et al., 2005
Plant and Yeast Cell Cultures Arabidopsis thaliana ecotypes Columbia and Ws and their mutant derivatives were used in this study. The conditions for plant growth and maintenance of Arabidopsis Columbia cell suspension culture have been described previously (Wen et al., 2006 ).
The haploid yeast strains used in this study are listed in Supplemental Table 1 online. Yeast cells were grown at 30°C in either rich YPD or in a synthetic dextrose (SD) medium (0.67% Bacto-yeast nitrogen base without amino acids, 2% glucose) supplemented with necessary nutrients as recommended (Sherman et al., 1983
Cloning Arabidopsis UEV1 cDNAs and Plasmid Construction
Yeast Two-Hybrid Analysis
Protein Expression, Purification, and GST Pull-Down Assay
Ub Conjugation Reaction
Yeast Killing and Spontaneous Mutagenesis Assay
Yeast strain DBY747 and its mms2
Expression Analysis by RNA Gel Blot and RT-PCR
For RT-PCR analysis, total RNA from various tissues was isolated using TRIzol and treated with DNaseI (Promega). Total RNA from mature pollen was extracted as described (Fei et al., 2004
Analysis of Microarray Expression Data
Seed Germination Assays
In Vitro Pollen Germination Assay
Accession Numbers
Supplemental Data
We thank Vipon Sawhney for the protocol on collecting pollen samples, Gordon Gray for the Arabidopsis cell suspension culture, Bernard Kunz for critical comments on the manuscript, and Michelle Hanna for proofreading the manuscript. This work was supported by Canadian Institutes of Health Research Operating Grant MOP-53240 to W.X. and a Natural Sciences and Engineering Research Council of Canada Discovery Grant to H.W.
1 Current address: Institute of Applied Genetics and Cell Biology, Department of Applied Plant Sciences and Plant Biotechnology, University of Natural Resources and Applied Life Sciences, Muthgasse 18, 1190 Vienna, Austria. 
2 Current address: Department of Biology, University of Pennsylvania, Philadelphia, PA 19104. The authors responsible for distribution of materials integral to the findings presented in this article in accordance with the policy described in the Instructions for Authors (www.plantcell.org) are: Hong Wang (hong.wang{at}usask.ca) and Wei Xiao (wei.xiao{at}usask.ca).
[W] Online version contains Web-only data. www.plantcell.org/cgi/doi/10.1105/tpc.107.051862 Received March 24, 2007; Revision received December 6, 2007. accepted December 17, 2007.
Andersen, P.L., Zhou, H., Pastushok, L., Moraes, T., McKenna, S., Ziola, B., Ellison, M.J., Dixit, V.M., and Xiao, W. (2005). Distinct regulation of Ubc13 functions by the two ubiquitin-conjugating enzyme variants Mms2 and Uev1A. J. Cell Biol. 170: 745–755. Arnason, T., and Ellison, M.J. (1994). Stress resistance in Saccharomyces cerevisiae is strongly correlated with assembly of a novel type of multiubiquitin chain. Mol. Cell. Biol. 14: 7876–7883. Bachmair, A., Novatchkova, M., Potuschak, T., and Eisenhaber, F. (2001). Ubiquitylation in plants: A post-genomic look at a post-translational modification. Trends Plant Sci. 6: 463–470.[CrossRef][Web of Science][Medline] Barbour, L., and Xiao, W. (2003). Regulation of alternative replication bypass pathways at stalled replication forks and its effects on genome stability: A yeast model. Mutat. Res. 532: 137–155.[Web of Science][Medline] Bartel, P.L., and Fields, S. (1995). Analyzing protein-protein interactions using two-hybrid system. Methods Enzymol. 254: 241–263.[Web of Science][Medline] Birnbaum, K., Shasha, D.E., Wang, J.Y., Jung, J.W., Lambert, G.M., Galbraith, D.W., and Benfey, P.N. (2003). A gene expression map of the Arabidopsis root. Science 302: 1956–1960. Blanc, G., Hokamp, K., and Wolfe, K.H. (2003). A recent polyploidy superimposed on older large-scale duplications in the Arabidopsis genome. Genome Res. 13: 137–144. Bothos, J., Summers, M.K., Venere, M., Scolnick, D.M., and Halazonetis, T.D. (2003). The Chfr mitotic checkpoint protein functions with Ubc13-Mms2 to form Lys-63-linked polyubiquitin chains. Oncogene 22: 7101–7107.[CrossRef][Web of Science][Medline] Broomfield, S., Chow, B.L., and Xiao, W. (1998). MMS2, encoding a ubiquitin-conjugating-enzyme-like protein, is a member of the yeast error-free postreplication repair pathway. Proc. Natl. Acad. Sci. USA 95: 5678–5683. Broomfield, S., Hryciw, T., and Xiao, W. (2001). DNA postreplication repair and mutagenesis in Saccharomyces cerevisiae. Mutat. Res. 486: 167–184.[Web of Science][Medline] Brown, M., Zhu, Y., Hemmingsen, S.M., and Xiao, W. (2002). Structural and functional conservation of error-free DNA postreplication repair in Schizosaccharomyces pombe. DNA Repair (Amst.) 1: 869–880.[CrossRef][Medline] Brusky, J., Zhu, Y., and Xiao, W. (2000). UBC13, a DNA-damage-inducible gene, is a member of the error-free postreplication repair pathway in Saccharomyces cerevisiae. Curr. Genet. 37: 168–174.[CrossRef][Web of Science][Medline] Craigon, D.J., James, N., Okyere, J., Higgins, J., Jotham, J., and May, S. (2004). NASCArrays: A repository for microarray data generated by NASC's transcriptomics service. Nucleic Acids Res. 32: D575–D577. Curtis, M.J., and Hays, J.B. (2007). Tolerance of dividing cells to replication stress in UVB-irradiated Arabidopsis roots: Requirements for DNA translesion polymerases Czechowski, T., Stitt, M., Altmann, T., Udvardi, M.K., and Scheible, W.R. (2005). Genome-wide identification and testing of superior reference genes for transcript normalization in Arabidopsis. Plant Physiol. 139: 5–17. Deng, L., Wang, C., Spencer, E., Yang, L., Braun, A., You, J., Slaughter, C., Pickart, C., and Chen, Z.J. (2000). Activation of the I Devoto, A., Muskett, P.R., and Shirasu, K. (2003). Role of ubiquitination in the regulation of plant defence against pathogens. Curr. Opin. Plant Biol. 6: 307–311.[CrossRef][Web of Science][Medline] Doss-Pepe, E.W., Chen, L., and Madura, K. (2005). Eddins, M.J., Carlile, C.M., Gomez, K.M., Pickart, C.M., and Wolberger, C. (2006). Mms2-Ubc13 covalently bound to ubiquitin reveals the structural basis of linkage-specific polyubiquitin chain formation. Nat. Struct. Mol. Biol. 13: 915–920.[CrossRef][Medline] Fan, L.M., Wang, Y.F., Wang, H., and Wu, W.H. (2001). In vitro Arabidopsis pollen germination and characterization of the inward potassium currents in Arabidopsis pollen grain protoplasts. J. Exp. Bot. 52: 1603–1614. Fei, H., Zhang, R., Pharis, R.P., and Sawhney, V.K. (2004). Pleiotropic effects of the male sterile33 (ms33) mutation in Arabidopsis are associated with modifications in endogenous gibberellins, indole-3-acetic acid and abscisic acid. Planta 219: 649–660.[Web of Science][Medline] Fields, S., and Song, O. (1989). A novel genetic system to detect protein-protein interactions. Nature 340: 245–246.[CrossRef][Medline] Fisk, H.A., and Yaffe, M.P. (1999). A role for ubiquitination in mitochondrial inheritance in Saccharomyces cerevisiae. J. Cell Biol. 145: 1199–1208. Franko, J., Ashley, C., and Xiao, W. (2001). Molecular cloning and functional characterization of two murine cDNAs which encode Ubc variants involved in DNA repair and mutagenesis. Biochim. Biophys. Acta 1519: 70–77.[Medline] Friedberg, E.C., Walker, G.C., Wolfram, S., Wood, R.D., Schultz, R.A., and Ellenberger, T. (2006). DNA Repair and Mutagenesis, 2nd ed. (Washington, DC: ASM Press). Friedman, W.E. (1999). Expression of the cell cycle in sperm of Arabidopsis: Implications for understanding patterns of gametogenesis and fertilization in plants and other eukaryotes. Development 126: 1065–1075.[Abstract] Galan, J.M., and Haguenauer-Tsapis, R. (1997). Ubiquitin lys63 is involved in ubiquitination of a yeast plasma membrane protein. EMBO J. 16: 5847–5854.[CrossRef][Web of Science][Medline] Garcia-Ortiz, M.V., Ariza, R.R., Hoffman, P.D., Hays, J.B., and Roldan-Arjona, T. (2004). Arabidopsis thaliana AtPOLK encodes a DinB-like DNA polymerase that extends mispaired primer termini and is highly expressed in a variety of tissues. Plant J. 39: 84–97.[CrossRef][Web of Science][Medline] Hall, T.A. (1999). BioEdit: A user-friendly biological sequence alignment editor and analysis program for Windows 95/98/NT. Nucleic Acids Symp. Ser. 41: 95–98. Hochstrasser, M. (1996). Ubiquitin-dependent protein degradation. Annu. Rev. Genet. 30: 405–439.[CrossRef][Web of Science][Medline] Hoege, C., Pfander, B., Moldovan, G.L., Pyrowolakis, G., and Jentsch, S. (2002). RAD6-dependent DNA repair is linked to modification of PCNA by ubiquitin and SUMO. Nature 419: 135–141.[CrossRef][Medline] Hofmann, R.M., and Pickart, C.M. (1999). Noncanonical MMS2-encoded ubiquitin-conjugating enzyme functions in assembly of novel polyubiquitin chains for DNA repair. Cell 96: 645–653.[CrossRef][Web of Science][Medline] Hofmann, R.M., and Pickart, C.M. (2001). In vitro assembly and recognition of Lys-63 polyubiquitin chains. J. Biol. Chem. 276: 27936–27943. Honys, D., and Twell, D. (2004). Transcriptome analysis of haploid male gametophyte development in Arabidopsis. Genome Biol. 5: R85.[CrossRef][Medline] Ito, H., Fukuda, Y., Murata, K., and Kimura, A. (1983). Transformation of intact yeast cells treated with alkali cations. J. Bacteriol. 153: 163–168. James, P., Halladay, J., and Craig, E.A. (1996). Genomic libraries and a host strain designed for highly efficient two-hybrid selection in yeast. Genetics 144: 1425–1436.[Abstract] Karsai, A., Muller, S., Platz, S., and Hauser, M.T. (2002). Evaluation of a homemade SYBR green I reaction mixture for real-time PCR quantification of gene expression. Biotechniques 32: 790–792, 794–796.[Web of Science][Medline] Laine, A., Topisirovic, I., Zhai, D., Reed, J.C., Borden, K.L., and Ronai, Z. (2006). Regulation of p53 localization and activity by Ubc13. Mol. Cell. Biol. 26: 8901–8913. McKenna, S., Moraes, T., Pastushok, L., Ptak, C., Xiao, W., Spyracopoulos, L., and Ellison, M.J. (2003). An NMR-based model of the ubiquitin-bound human ubiquitin conjugation complex Mms2.Ubc13. The structural basis for lysine 63 chain catalysis. J. Biol. Chem. 278: 13151–13158. McKenna, S., Spyracopoulos, L., Moraes, T., Pastushok, L., Ptak, C., Xiao, W., and Ellison, M.J. (2001). Noncovalent interaction between ubiquitin and the human DNA repair protein Mms2 is required for Ubc13-mediated polyubiquitination. J. Biol. Chem. 276: 40120–40126. Moon, J., Parry, G., and Estelle, M. (2004). The ubiquitin-proteasome pathway and plant development. Plant Cell 16: 3181–3195. Muralidhar, M.G., and Thomas, J.B. (1993). The Drosophila bendless gene encodes a neural protein related to ubiquitin-conjugating enzymes. Neuron 11: 253–266.[CrossRef][Web of Science][Medline] Nawy, T., Lee, J.Y., Colinas, J., Wang, J.Y., Thongrod, S.C., Malamy, J.E., Birnbaum, K., and Benfey, P.N. (2005). Transcriptional profile of the Arabidopsis root quiescent center. Plant Cell 17: 1908–1925. Oh, C.E., McMahon, R., Benzer, S., and Tanouye, M.A. (1994). bendless, a Drosophila gene affecting neuronal connectivity, encodes a ubiquitin-conjugating enzyme homolog. J. Neurosci. 14: 3166–3179.[Abstract] Papouli, E., Chen, S., Davies, A.A., Huttner, D., Krejci, L., Sung, P., and Ulrich, H.D. (2005). Crosstalk between SUMO and ubiquitin on PCNA is mediated by recruitment of the helicase Srs2p. Mol. Cell 19: 123–133.[CrossRef][Web of Science][Medline] Pastushok, L., Moraes, T.F., Ellison, M.J., and Xiao, W. (2005). A single Mms2 "key" residue insertion into a Ubc13 pocket determines the interface specificity of a human Lys-63 ubiquitin conjugation complex. J. Biol. Chem. 280: 17891–17900. Pastushok, L., Spyracopoulos, L., and Xiao, W. (2007). Two Mms2 residues that cooperatively interact with ubiquitin are critical for Lys-63-linked polyubiquitination. FEBS Lett. 581: 5343–5348.[Medline] Pastushok, L., and Xiao, W. (2004). DNA postreplication repair modulated by ubiquitination and sumoylation. Adv. Protein Chem. 69: 279–306.[Medline] Pfander, B., Moldovan, G.L., Sacher, M., Hoege, C., and Jentsch, S. (2005). SUMO-modified PCNA recruits Srs2 to prevent recombination during S phase. Nature 436: 428–433.[Medline] Pickart, C.M. (2001a). Ubiquitin enters the new millennium. Mol. Cell 8: 499–504.[CrossRef][Web of Science][Medline] Pickart, C.M. (2001b). Mechanisms underlying ubiquitination. Annu. Rev. Biochem. 70: 503–533.[CrossRef][Web of Science][Medline] Sakamoto, A., Lan, V.T., Hase, Y., Shikazono, N., Matsunaga, T., and Tanaka, A. (2003). Disruption of the AtREV3 gene causes hypersensitivity to ultraviolet B light and Sancho, E., et al. (1998). Role of UEV-1, an inactive variant of the E2 ubiquitin-conjugating enzymes, in in vitro differentiation and cell cycle behavior of HT-29-M6 intestinal mucosecretory cells. Mol. Cell. Biol. 18: 576–589. Schmid, M., Davison, T.S., Henz, S.R., Pape, U.J., Demar, M., Vingron, M., Scholkopf, B., Weigel, D., and Lohmann, J.U. (2005). A gene expression map of Arabidopsis thaliana development. Nat. Genet. 37: 501–506.[CrossRef][Web of Science][Medline] Schmid, M., Uhlenhaut, N.H., Godard, F., Demar, M., Bressan, R., Weigel, D., and Lohmann, J.U. (2003). Dissection of floral induction pathways using global expression analysis. Development 130: 6001–6012. Sherman, F., Fink, G.R., and Hicks, J. (1983). Methods in Yeast Genetics. (Cold Spring Harbor, NY: Cold Spring Harbor Laboratory Press). Smyth, D.R., Bowman, J.L., and Meyerowitz, E.M. (1990). Early flower development in Arabidopsis. Plant Cell 2: 755–767. Spence, J., Gali, R.R., Dittmar, G., Sherman, F., Karin, M., and Finley, D. (2000). Cell cycle-regulated modification of the ribosome by a variant multiubiquitin chain. Cell 102: 67–76.[CrossRef][Web of Science][Medline] Stelter, P., and Ulrich, H.D. (2003). Control of spontaneous and damage-induced mutagenesis by SUMO and ubiquitin conjugation. Nature 425: 188–191.[CrossRef][Medline] Sturn, A., Quackenbush, J., and Trajanoski, Z. (2002). Genesis: Cluster analysis of microarray data. Bioinformatics 18: 207–208. Suzuki, G., Yanagawa, Y., Kwok, S.F., Matsui, M., and Deng, X.W. (2002). Arabidopsis COP10 is a ubiquitin-conjugating enzyme variant that acts together with COP1 and the COP9 signalosome in repressing photomorphogenesis. Genes Dev. 16: 554–559. Takahashi, S., Sakamoto, A., Sato, S., Kato, T., Tabata, S., and Tanaka, A. (2005). Roles of Arabidopsis AtREV1 and AtREV7 in translesion synthesis. Plant Physiol. 138: 870–881. Tsui, C., Raguraj, A., and Pickart, C.M. (2005). Ubiquitin binding site of the ubiquitin E2 variant (UEV) protein Mms2 is required for DNA damage tolerance in the yeast RAD6 pathway. J. Biol. Chem. 280: 19829–19835. Tuteja, N., Singh, M.B., Misra, M.K., Bhalla, P.L., and Tuteja, R. (2001). Molecular mechanisms of DNA damage and repair: Progress in plants. Crit. Rev. Biochem. Mol. Biol. 36: 337–397.[CrossRef][Web of Science][Medline] Vicient, C.M., and Delseny, M. (1999). Isolation of total RNA from Arabidopsis thaliana seeds. Anal. Biochem. 268: 412–413.[CrossRef][Web of Science][Medline] Vierstra, R.D. (2003). The ubiquitin/26S proteasome pathway, the complex last chapter in the life of many plant proteins. Trends Plant Sci. 8: 135–142.[CrossRef][Web of Science][Medline] Villalobo, E., Morin, L., Moch, C., Lescasse, R., Hanna, M., Xiao, W., and Baroin-Tourancheau, A. (2002). A homologue of CROC-1 in a ciliated protist (Sterkiella histriomuscorum) testifies to the ancient origin of the ubiquitin-conjugating enzyme variant family. Mol. Biol. Evol. 19: 39–48. Wang, C., Deng, L., Hong, M., Akkaraju, G.R., Inoue, J., and Chen, Z.J. (2001). TAK1 is a ubiquitin-dependent kinase of MKK and IKK. Nature 412: 346–351.[CrossRef][Medline] Wang, H., Datla, R., Georges, F., Loewen, M., and Cutler, A.J. (1995). Promoters from kin1 and cor6.6, two homologous Arabidopsis thaliana genes: Transcriptional regulation and gene expression induced by low temperature, ABA, osmoticum and dehydration. Plant Mol. Biol. 28: 605–617.[CrossRef][Web of Science][Medline] Wen, R., Newton, L., Li, G., Wang, H., and Xiao, W. (2006). Arabidopsis thaliana UBC13: Implication of error-free DNA damage tolerance and Lys-63-linked polyubiquitylation in plants. Plant Mol. Biol. 61: 241–253.[CrossRef][Web of Science][Medline] Xiao, W., Chow, B.L., Fontanie, T., Ma, L., Bacchetti, S., Hryciw, T., and Broomfield, S. (1999). Genetic interactions between error-prone and error-free postreplication repair pathways in Saccharomyces cerevisiae. Mutat. Res. 435: 1–11.[Web of Science][Medline] Xiao, W., Lin, S.L., Broomfield, S., Chow, B.L., and Wei, Y.F. (1998). The products of the yeast MMS2 and two human homologs (hMMS2 and CROC-1) define a structurally and functionally conserved Ubc-like protein family. Nucleic Acids Res. 26: 3908–3914. Xiao, W., and Samson, L. (1993). In vivo evidence for endogenous DNA alkylation damage as a source of spontaneous mutation in eukaryotic cells. Proc. Natl. Acad. Sci. USA 90: 2117–2121. Yamamoto, M., et al. (2006a). Key function for the Ubc13 E2 ubiquitin-conjugating enzyme in immune receptor signaling. Nat. Immunol. 7: 962–970.[CrossRef][Web of Science][Medline] Yamamoto, M., Sato, S., Saitoh, T., Sakurai, H., Uematsu, S., Kawai, T., Ishii, K.J., Takeuchi, O., and Akira, S. (2006b). Cutting edge: Pivotal function of Ubc13 in thymocyte TCR signaling. J. Immunol. 177: 7520–7524. Zhou, H., Wertz, I., O'Rourke, K., Ultsch, M., Seshagiri, S., Eby, M., Xiao, W., and Dixit, V.M. (2004). Bcl10 activates the NF- This article has been cited by other articles:
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INTRODUCTION
) transformants were grown overnight and printed onto YPD and YPD + 0.025% MMS gradient plates. The plates were incubated at 30°C for 2 d before being photographed. The arrow indicates higher MMS concentrations. Several transformants of each treatment were tested with the same result, and only one is shown here.
-Synuclein and parkin contribute to the assembly of ubiquitin lysine 63-linked multiubiquitin chains. J. Biol. Chem. 280: 16619–16624.
