Characterization of four rice UEV1 genes required for Lys63-linked polyubiquitination and distinct functions
© The Author(s). 2017
Received: 23 March 2017
Accepted: 3 July 2017
Published: 17 July 2017
The error-free branch of the DNA-damage tolerance (DDT) pathway is orchestrated by Lys63-linked polyubiquitination of proliferating cell nuclear antigen (PCNA), and this polyubiquitination is mediated by a Ubc13-Uev complex in yeast. We have previously cloned OsUBC13 from rice, whose product functions as an E2 to promote Lys63-linked ubiquitin chain assembly in the presence of yeast or human Uev.
Here we identify four highly conserved UEV1 genes in rice whose products are able to form stable heterodimers with OsUbc13 and mediate Lys63-linked ubiquitin chain assembly. Expression of OsUEV1s is able to rescue the yeast mms2 mutant from death caused by DNA-damaging agents. Interestingly, OsUev1A contains a unique C-terminal tail with a conserved prenylation site not found in the other three OsUev1s, and this post-translational modification appears to be required for its unique subcellular distribution and association with the membrane. The analysis of OsUEV1 expression profiles obtained from the Genevestigator database indicates that these genes are differentially regulated.
We speculate that different OsUev1s play distinct roles by serving as a regulatory subunit of the Ubc13-Uev1 complex to respond to diverse cellular, developmental and environmental signals.
Ubiquitination is a critical post-translational protein modification process in eukaryotic cells, which involves a small protein modifier named ubiquitin (Ub). Although ubiquitination is well known to target proteins for degradation [1, 2], several non-proteolytic roles have also been found including manipulating protein interaction, activities and localization [3–5]. Different fates of the target protein after ubiquitination are often dictated by whether it is monoubiquitinated, or additional ubiquitins are attached to form a poly-Ub chain. In the latter case, the C-terminus of an incoming Ub can be linked to one of seven surface lysine residues (Lys6, Lys11, Lys27, Lys29, Lys33, Lys48 and Lys63) on the previous Ub [3, 6]. It was found that different poly-Ub chains have different topological and chemical properties; for example, while Lys11, Lys29 and Lys48 linked chains lead to protein degradation [2, 3, 7], the Lys63-linked chain is generally involved in signal transduction .
Ubiquitination was initially implicated in DNA-damage response when Rad6, an E2 enzyme, was found to be required for post-replication repair (PRR) in budding yeast . Rad6, along with its cognate E3 Rad18, monoubiquitinates proliferating cell nuclear antigen (PCNA) at the Lys164 residue in response to replication-blocking DNA damage; this monoubiquitination leads to translesion DNA synthesis (TLS). The monoubiquitinated PCNA can be further polyubiquitinated at the same residue by the E2-E3 complex Mms2-Ubc13-Rad5 [9, 10], which is required for error-free lesion bypass [11–14] via template switch [15, 16]. This process appears to be conserved in eukaryotic organisms from yeast to human, and is named DNA-damage tolerance (DDT) [17, 18].
Owing to their sessile nature, plants are continuously under different types of stresses, such as DNA damage by UV exposure. These stresses severely compromise plant survival, reduce crop yield and threaten food security. Plants have established several strategies to cope with DNA-damage stresses, including various DNA repair pathways and tolerance of replication blocks by efficient TLS polymerases [19–24]. Meanwhile a few reports also indicate the conservation of error-free DDT in Arabidopsis [25–27]; however, little is known about the underlying mechanisms. We previously reported the cloning and characterization of rice UBC13, a putative error-free DDT gene, and showed that it is able to functionally complement the corresponding yeast ubc13 mutant’s defect in PRR, and its product mediates Lys63-linked polyubiquitination in vitro . In both cases, rice Ubc13 has to rely on a heterologous Ubc-E2 variant (Uev). Indeed, Ubc13 and Uev proteins from yeast or mammalian cells form a stable heterodimer, which is absolutely required for Lys63-linked poly-Ub chain assembly [29–31], and this process appears to be highly conserved in eukaryotes . In this study, four rice UEV1 genes are identified and functionally characterized. Interestingly, one of the four rice UEV1 products, Uev1A, is deemed to be post-translationally modified in its C-terminus, which makes it functionally different from other three Uev1s, suggesting that they are involved in multiple cellular processes, that they have distinct functions and that rice Uevs may serve as a regulatory subunit to modulate Ubc13 activities.
The rice genome encodes four highly conserved UEV1 genes
Our previous work has identified the UBC13 gene in rice, which is predicted to produce a protein which is highly conserved with Ubc13s from some other species . In general, Ubc13 works with Uev as a heterodimer to catalyze the assembling of Lys63-linked Ub chains, and OsUbc13 was proved to be able to interact with Uevs from yeast and human to achieve this goal. Therefore, it is reasonable to predict that the rice genome contains its own conserved UEV gene(s). In this study, the Arabidopsis UEV1A gene was used to BLAST the rice genome in the Rice Annotation Project Database (RAP-DB, http://rapdb.dna.affrc.go.jp/index.html). Four genes were retrieved and named OsUEV1A (Os03g0712300, GenBank accession number XM_015777395.1), OsUEV1B (Os12g0605400, XM_015764791.1), OsUEV1C (Os09g0297100, XM_015756494.1) and OsUEV1D (Os04g0684800, XM_015780422.1). The exon-intron organization and coding sequences of these rice loci were determined through sequence comparison with the PCR-amplified corresponding full-length cDNAs and available sequences from RAP-DB. Based on the cDNA PCR products detected, all the OsUEV1 cDNA products were identical to corresponding annotations on RAP-DB.
Based on the ORF sequences of all four UEV1 genes from rice, OsUEV1B, OsUEV1C and OsUEV1D are predicted to encode proteins with 146, 148 and 147 amino acids, respectively, whereas the predicted OsUev1A protein contains 161 amino acids with a C-terminal extension, which was also found in AtUev1A, AtUev1B and BdUev1A (Fig. 1b). It was noted that all plant Uev1s with the C-terminal extension contain a conserved CaaX motif predicted to be a target of prenylation, a protein lipid modification that facilitates the protein-protein or protein-membrane interaction by attaching the isoprenoid groups (a 15-carbon farnesyl or 20-carbon geranlygeranyl) to the Cys residue (blue asterisk) [33, 34]. In addition, several critical residues implicated in Uev activity are also conserved among all Uev1s, including hMms2-F13 (red asterisk) known to be required for the physical interaction with Ubc13 , and hMms2-S32 and I62 (green asterisks) required for non-covalent interaction with Ub and poly-Ub chain assembling [35, 36] (Fig. 1b).
OsUev1s physically interact with OsUbc13 to form a stable heterodimer
To further confirm the direct interaction between OsUbc13 and OsUev1s, we performed an in vitro glutathione S-transferase (GST) pull-down assay with recombinant proteins purified from Escherichia coli. Indeed, all GST-tagged recombinant OsUev1s are able to pull-down His6-tagged OsUbc13, while as a control, GST protein alone fails to do so (Fig. 2b). From the above observations, we conclude that all four OsUev1s are able to interact with OsUbc13 directly and form stable heterodimers.
OsUev1s are required for Lys63-linked polyubiquitin chain assembly in vitro by OsUbc13
Functional complementation of yeast mms2 by OsUEV1 genes
Since OsUBC13 is also able to complement the yeast ubc13 null mutant , we next asked whether the OsUbc13-OsUev1 complexes are able to functionally complement the yeast mms2 ubc13 double mutant. The yeast mms2 ubc13 double mutant cells were co-transformed with two yeast plasmids expressing OsUBC13 and OsUEV1, or a corresponding empty vector. As expected, neither OsUBC13 nor OsUEV1s with corresponding empty vectors is able to rescue the yeast mms2 ubc13 double mutant. Surprisingly, while the combination of OsUBC13 with OsUEV1B, OsUEV1C or OsUEV1D restored the yeast mms2 ubc13 double mutant sensitivity to MMS to the wild-type level, the combination of OsUBC13 and OsUEV1A did not provide mms2 ubc13 mutant cells with MMS resistance (Fig. 4b).
Roles of the OsUev1A C-terminal domain and its putative prenylation site
The C-terminal tail and putative prenylation of OsUev1A determines its subcellular localization and membrane association
Since protein prenylation has been reported to facilitate protein-protein interaction and/or protein-membrane interaction [33, 34], we asked whether GFP-tagged OsUev1 variants are indeed associated with the membrane. Tobacco leaves transformed with GFP-tagged OsUev1 variants were subject to a Triton X-114 based protein-partitioning assay. As shown in Fig. 6b, GFP-tagged OsUev1A is almost exclusively found in the detergent (D) phase, whereas C-terminally truncated GFP-OsUev1A is partially diffused to the aqueous (A) phase. The partial dissociation of Os-Uev1A-∆CT from membrane fraction is because either it still contains another membrane association motif, or the ectopically expressed GFP-tagged OsUev1 level is higher than the native Uev1A. Nevertheless, these results collectively indicate that OsUev1A preferentially associates with membrane and that this association is dependent on its C-terminal sequence and probably on its prenylation, whereas other three OsUev1s are soluble proteins spread in both cytoplasm and the nucleus.
Expression of OsUEV1s in different tissues and during different developmental stages
In this study, we identified and cloned four highly conserved UEV genes from the rice genome and our in vitro studies confirm that these Uevs are able to interact with OsUbc13 to form a stable heterodimer and mediate Lys63-linked polyubiquitination. Functional studies indicate that these rice UEV genes can restore cellular activity of the yeast mms2 null mutant for resistance to a DNA-damaging agent, reminiscent of the ability of OsUBC13 to restore the corresponding yeast ubc13 mutant . Furthermore, several observations are consistent with the notion that the four OsUEV1s confer different functions in vivo. Firstly, when both yeast MMS2 and UBC13 genes are replaced by different combinations of OsUBC13 and OsUEV1s, three of them can fully restore the DNA-damage tolerance activity, while OsUBC13-OsUEV1A cannot. Secondly, in a yeast two-hybrid assay the OsUbc13-OsUev1A interaction appears to be stronger than the other three pairs, ruling out a possibility that the lack of functional complementation by OsUBC13-OsUEV1A is due to reduced physical interaction. Finally, the subcellular localization of OsUev1A differs from that of OsUev1D (and presumably OsUev1B and OsUev1C) in plants. While OsUev1D behaves like a small soluble protein and appears to be enriched in the nucleus, OsUev1A is excluded from the nucleus and appears to be membrane-bound. The above observations collectively indicate that OsUev1s confer function(s) in addition to DDT and that different OsUev1s may have distinct physiological functions. This conclusion is not unexpected as in yeast, the regulation of the DDT pathway is the only known function of Ubc13-Mms2; however, the two distinct Ubc13-Uev complexes turn out to be multi-functional in multi-cellular organisms like mammals . Hence, it is reasonable to speculate that Uevs are also multi-functional in plants. Indeed, plant Ubc13 has been implicated to function in apical dominance , iron metabolism , innate immunity  and auxin signaling , and at least some of the above functions may require the Ubc13-Uev E2 complex and Lys63-linked ubiquitination.
Mammalian genomes contain one UBC13 gene and at least two UEV genes, and the UEV genes often confer distinct functions. For example, mammalian Ubc13 regulates the DDT pathway by interacting with Mms2 and mediates NF-κB signaling by associating with Uev1A . In this study, we identified four OsUEV genes in rice and at least one of them, OsUEV1A, functions differently from other OsUEV genes. Similar results are also observed in Arabidopsis, in which AtUev1A and AtUev1B contain additional C-terminal sequences . A novel finding in this study is that OsUev1A distributes differently in the cellular compartment than other OsUev1s, and that its unique localization and membrane-bound property can be abolished by removal of the C-terminus or simply mutating the predicted prenylation site. Although exactly which cellular role(s) it plays remains unknown, it can be cautiously predicted based on this study that it is membrane-related and non-nuclear. This function must be critical for the plant development and/or environmental response, as essentially all known plant genomes contain at least one OsUev1A ortholog with predicted CaaX motif at their C-terminus (data not shown). On the other hand, the remaining three OsUev1s may function in DNA-damage response like their Arabidopsis Uev1D counterparts , consistent with the observed OsUev1D nuclear localization. Given that AtUbc13 has been implicated in several cellular processes and these functions are likely conserved with OsUbc13, it is of great interest to investigate which Ubc13-mediated cellular process involves which OsUev1 and to discover additional cellular processes in which Ubc13-Uev participates.
As a non-canonical ubiquitination, Lys63-linked ubiquitination is most likely involved in stress response signaling, where Ubc13-Uev plays a critical role in assembling Lys63-linked poly-Ub chains on the target protein. Therefore, it is conceivable that its activity is tightly regulated in response to different environmental signals. To date, no report has found altered activity of Ubc13 in plant species examined [25, 28, 43]. Instead, its activity and specificity are largely determined by the cognate Uev, and the cellular levels of Uev appear to fluctuate in different tissues and in response to various environmental stresses [44–46]. Furthermore, the pathway involvement of Lys63-linked Ub chain is mainly determined by the Uev that interacts with Ubc13 . In this study, four distinct UEV genes in rice also display different expression patterns among different tissues, life stages and environmental stresses. In addition to the constitutively expressed OsUEV1B and OsUEV1C genes, the OsUEV1A and OsUEV1D expression fluctuates under all the above conditions, suggesting that these two gene products play regulatory roles under different biological processes. Hence, the regulation of Uev activity appears to be evolutionarily preferred and Uevs serve as regulatory subunits of the Ubc13-Uev E2 complex in response to distinct cellular and environmental signals.
In this article, we report the molecular cloning and functional characterization of four rice UEV1 genes. Like other plant species, rice also contains two classes of UEV1 genes with their encoded proteins differ in the C-terminal extension. This study reveals that OsUev1A contains a C-terminal tail not found in other three OsUev1s, that the tail sequences are highly conserved within higher plants, from both monocotyledon and dicotyledon, and that a putative posttranslational modification site is also conserved. Our limited experimental results showed that the two classes of OsUEV1s genes function differently in a heterologous yeast host and that their protein subcellular distribution patterns are also different in plants. Furthermore, the above differences are attributed to the OsUev1A C-terminal tail and most likely to its putative prenylation. Unlike the OsUBC13 gene that is constitutively expressed, database analyses reveal that the expression of four OsUEV1 genes fluctuates dramatically in different tissues, during different developmental stages as well as in response to various biotic and abiotic stresses, suggesting that these OsUEV1 gene products regulate the Ubc13-Uev1 activity.
Plant materials and yeast cell culture
Rice (Oryza sativa L. cv. Japonica) seeds were surface sterilized with 2% NaClO for 30 min after a pre-wash by sterile distilled water, followed by washing seven times in sterile water. The sterilized rice seeds were plated in Murashige and Skoog (MS) plates containing 2.2 g/l minimal organics, 10 g/l sucrose and 1% agar. They were cultured in a growth chamber (16 h light/8 h dark and 30 °C).
Yeast strains used in this study include PJ69-4A  for the yeast two-hybrid assay, HK578-10D (MATa ade2–1 can1–100 his3–11,15 leu2–3, 112 trp1–1 ura3–1) and its mms2Δ::HIS3 derivative WXY902 and mms2Δ::HIS3 ubc13Δ::hisG-URA3-hisG derivative WXY955 for the functional analysis. Yeast cells were grown at 30 °C in either rich YPD or a synthetic dextrose (SD) medium supplemented with nutrients as instructed . To make plates, 2% agar was added to YPD or SD medium prior to autoclaving. Yeast cells were transformed by a LiAc method .
Cloning rice UEV1 cDNAs and plasmid construction
To clone the full-length OsUEV1 open reading frames (ORFs), total RNA was extracted from rice seedlings with TRIzol reagents (Invitrogen, Carlsbad), which was used as a template for RT-PCR using the RevertAid First Strand cDNA Synthesis Kit (Fermentas). Gene-specific primers are as follows: OsUEV1A: 5′-taaccggaattcATGGGGTCCGAGGGATC-3′ and 5′-ggcacgcgtcgacTTACATGATGACACACCTA-3′; OsUEV1A-C158S: 5′-ggcacgcgtcgacTTACATGATGACACTCCTA-3′; OsUEV1A-∆CT: 5′-ggcacgcgtcgacTTAGCCATCATGGGGTTGATG-3′; OsUEV1B: 5′-gaaccggaattcATGGCGTCGAGTGGAGAT-3′ and 5′-gcacgcgtcgacCTAGAAGAATGTCCCCTC-3′; OsUEV1C: 5′-tgaccggaattcATGACGCTGGGGAGCTC-3′ and 5′-gcacgcgtcgacCTAGAAGAACGTCCCTTC-3′; OsUEV1D: 5′-taactggaattcATGACGATCGGCGGCG-3′ and 5′-tcccgcgtcgacCTAGAAGAAGGTCCCTTC-3′. The forward primers contain the EcoRI restriction site and the reverse primers contain the SalI site, as italicized. The PCR product of OsUEV1A-1D ORFs were cloned into a yeast two-hybrid vector pGAD424Bg, which was derived from pGAD424 .
Yeast two-hybrid analysis
Recombinant protein purification and ubiquitination assay
OsUEV1 ORFs were isolated from pGAD-OsUev1s and cloned into pGEX6p-1. The resulting pGEX-OsUev1s were transformed into E. coli BL21 CodonPlus (DE3)-RIL cells. The pGEX-OsUev1 fusion proteins were purified following a previously published protocol . Meanwhile, GST and His6-OsUbc13 were produced and purified as previously described . For an in vitro ubiquitination assay, a previously described protocol  was followed.
GST pull-down assay
The E. coli BL21 competent cells were transformed with either pGEX6p-1, pGEX-OsUev1s alone, or co-transformed with pET-OsUbc13. The whole-cell extracts were incubated with Glutathione Sepharose 4B Microspin™ beads (17–0756-01, GE Healthcare) at 4 °C for 2 h, which were then harvested by centrifugation, washed 5 times with a lysis buffer and boiled with 2 × loading buffer. The products were analyzed on a 12% SDS-PAGE gel.
Yeast gradient plate assay
Yeast strain HK578-10D and its isogenic mms2∆ single or ubc13∆ mms2∆ double mutants were either singly transformed with pGAD-OsUev1A-1D or co-transformed with pGAD-OsUev1s and pGBT-OsUbc13. The transformants were selected on SD-Leu (for mms2∆) or SD-Leu-Trp (for ubc13∆ mms2∆) plates. The gradient plate assay was conducted as described .
The ORFs of OsUEV1s and derivatives were amplified and cloned into the pCAMBIA1302 vector containing an N-terminal GFP tag. These GFP-OsUev1s constructs were transformed into the Agrobacterium tumefaciens (GV3101/pMP90), and positive colonies were cultured overnight and infiltrated into Nicotiana benthamiana leaves as described . After 2–3 day incubation, epidermal cells of the transformed tobacco leaf were viewed by confocal microscopy (Zeiss LSM 780, Germany). Excitation parameters are 488 nm and 405 nm for GFP and DAPI, respectively.
Protein partitioning assay
For protein partitioning assay, total protein was extracted from transformed N. benthamiana leaves using a buffer containing 50 mM Tris-HCl pH 8.0, 0.3 M NaCl, 1% TritonX-114, 10 mM PMSF, 3 mM DDT and 1 tablet (for 50 ml buffer) protease inhibitors (Roche). The extract was incubated in Triton X-114 containing buffer for 1 h at 4 °C before centrifugation at 12,000 g for 10 min at 4 °C. Samples were then incubated at 37 °C for 5 min and centrifuged at 12,000 g. The aqueous upper phase and detergent-enriched lower phase were separated and extracted once again with detergent and aqueous solutions, respectively. The resulting four samples were adjusted to equal volume and proteins were precipitated with chloroform/methanol prior to Western blot analysis.
The authors wish to thank Guang Yang for technical assistance and Michelle Hanna for proofreading the manuscript.
This work is supported by the National Natural Science Foundation of China (31270823) and Beijing Municipal Commission of Education to WX.
Availability of data materials
The datasets acquired and/or analyzed during the current study are available from the corresponding author on reasonable request.
QW, YZ and WX conceived and designed experiments; QW, YZ and XZ performed experiments. QW, YZ and WX analyzed data and wrote the article. All authors read and approved the final manuscript.
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- Glickman MH, Ciechanover A. The ubiquitin-proteasome proteolytic pathway: destruction for the sake of construction. Physiol Rev. 2002;82(2):373–428.View ArticlePubMedGoogle Scholar
- Hershko A, Ciechanover A. The ubiquitin system. Annu Rev Biochem. 1998;67:425–79.View ArticlePubMedGoogle Scholar
- Komander D, Rape M. The ubiquitin code. Annu Rev Biochem. 2012;81:203–29.View ArticlePubMedGoogle Scholar
- Jackson SP, Durocher D. Regulation of DNA damage responses by ubiquitin and SUMO. Mol Cell. 2013;49(5):795–807.View ArticlePubMedGoogle Scholar
- Chen ZJ, Sun LJ. Nonproteolytic functions of ubiquitin in cell signaling. Mol Cell. 2009;33(3):275–86.View ArticlePubMedGoogle Scholar
- Komander D. The emerging complexity of protein ubiquitination. Biochem Soc Trans. 2009;37(Pt 5):937–53.View ArticlePubMedGoogle Scholar
- Wickliffe KE, Williamson A, Meyer HJ, Kelly A, Rape M. K11-linked ubiquitin chains as novel regulators of cell division. Trends Cell Biol. 2011;21(11):656–63.View ArticlePubMedPubMed CentralGoogle Scholar
- Jentsch S, McGrath JP, Varshavsky A. The yeast DNA repair gene RAD6 encodes a ubiquitin-conjugating enzyme. Nature. 1987;329(6135):131–4.View ArticlePubMedGoogle Scholar
- Hoege C, Pfander B, Moldovan GL, Pyrowolakis G, Jentsch S. RAD6-dependent DNA repair is linked to modification of PCNA by ubiquitin and SUMO. Nature. 2002;419(6903):135–41.View ArticlePubMedGoogle Scholar
- Stelter P, Ulrich HD. Control of spontaneous and damage-induced mutagenesis by SUMO and ubiquitin conjugation. Nature. 2003;425(6954):188–91.View ArticlePubMedGoogle Scholar
- Broomfield S, Chow BL, Xiao W. MMS2, encoding a ubiquitin-conjugating-enzyme-like protein, is a member of the yeast error-free postreplication repair pathway. Proc Natl Acad Sci U S A. 1998;95(10):5678–83.View ArticlePubMedPubMed CentralGoogle Scholar
- Brusky J, Zhu Y, Xiao W. UBC13, a DNA-damage-inducible gene, is a member of the error-free postreplication repair pathway in Saccharomyces cerevisiae. Curr Genet. 2000;37(3):168–74.View ArticlePubMedGoogle Scholar
- Xiao W, Chow BL, Broomfield S, Hanna M. The Saccharomyces cerevisiae RAD6 group is composed of an error-prone and two error-free postreplication repair pathways. Genetics. 2000;155(4):1633–41.PubMedPubMed CentralGoogle Scholar
- Ulrich HD, Jentsch S. Two RING finger proteins mediate cooperation between ubiquitin-conjugating enzymes in DNA repair. EMBO J. 2000;19(13):3388–97.View ArticlePubMedPubMed CentralGoogle Scholar
- Xu X, Blackwell S, Lin A, Li F, Qin Z, Xiao W. Error-free DNA-damage tolerance in Saccharomyces cerevisiae. Mutat Res Rev Mutat Res. 2015;764:43–50.View ArticlePubMedGoogle Scholar
- Ball LG, Zhang K, Cobb JA, Boone C, Xiao W. The yeast Shu complex couples error-free post-replication repair to homologous recombination. Mol Microbiol. 2009;73(1):89–102.View ArticlePubMedGoogle Scholar
- Andersen PL, Xu F, Xiao W. Eukaryotic DNA damage tolerance and translesion synthesis through covalent modifications of PCNA. Cell Res. 2008;18(1):162–73.View ArticlePubMedGoogle Scholar
- Pastushok L, Xiao W. DNA postreplication repair modulated by ubiquitination and sumoylation. Adv Protein Chem. 2004;69:279–306.View ArticlePubMedGoogle Scholar
- Curtis MJ, Hays JB. Tolerance of dividing cells to replication stress in UVB-irradiated Arabidopsis roots: requirements for DNA translesion polymerases eta and zeta. DNA Repair (Amst). 2007;6(9):1341–58.View ArticlePubMedGoogle Scholar
- Anderson HJ, Vonarx EJ, Pastushok L, Nakagawa M, Katafuchi A, Gruz P, Di Rubbo A, Grice DM, Osmond MJ, Sakamoto AN, et al. Arabidopsis thaliana Y-family DNA polymerase eta catalyses translesion synthesis and interacts functionally with PCNA2. Plant J. 2008;55(6):895–908.View ArticlePubMedGoogle Scholar
- Garcia-Ortiz MV, Ariza RR, Hoffman PD, Hays JB, Roldan-Arjona T. 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. 2004;39(1):84–97.View ArticlePubMedGoogle Scholar
- Sakamoto A, Lan VT, Hase Y, Shikazono N, Matsunaga T, Tanaka A. Disruption of the AtREV3 gene causes hypersensitivity to ultraviolet B light and gamma-rays in Arabidopsis: implication of the presence of a translesion synthesis mechanism in plants. Plant Cell. 2003;15(9):2042–57.View ArticlePubMedPubMed CentralGoogle Scholar
- Takahashi S, Sakamoto A, Sato S, Kato T, Tabata S, Tanaka A. Roles of Arabidopsis AtREV1 and AtREV7 in translesion synthesis. Plant Physiol. 2005;138(2):870–81.View ArticlePubMedPubMed CentralGoogle Scholar
- Kunz BA, Xiao W. DNA damage tolerance in plants via translesion synthesis. Genes, Genomes & Genomics. 2007;1(1):89–99.Google Scholar
- Wen R, Newton L, Li G, Wang H, Xiao W. Arabidopsis thaliana UBC13: implication of error-free DNA damage tolerance and Lys63-linked polyubiquitylation in plants. Plant Mol Biol. 2006;61(1–2):241–53.View ArticlePubMedGoogle Scholar
- Wen R, Torres-Acosta JA, Pastushok L, Lai XQ, Pelzer L, Wang H, Xiao W. Arabidopsis UEV1D promotes lysine-63-linked polyubiquitination and is involved in DNA damage response. Plant Cell. 2008;20(1):213–27.View ArticlePubMedPubMed CentralGoogle Scholar
- Wang S, Wen R, Shi X, Lambrecht A, Wang H, Xiao W. RAD5a and REV3 function in two alternative pathways of DNA-damage tolerance in Arabidopsis. DNA Repair. 2011;10(6):620–8.View ArticlePubMedGoogle Scholar
- Zang YP, Wang Q, Xue CY, Li MN, Wen R, Xiao W. Rice UBC13, a candidate housekeeping gene, is required for K63-linked polyubiquitination and tolerance to DNA damage. Rice. 2012;5:24.View ArticlePubMedGoogle Scholar
- Pastushok L, Moraes TF, Ellison MJ, Xiao W. A single Mms2 "key" residue insertion into a Ubc13 pocket determines the interface specificity of a human Lys63 ubiquitin conjugation complex. J Biol Chem. 2005;280(18):17891–900.View ArticlePubMedGoogle Scholar
- McKenna S, Spyracopoulos L, Moraes T, Pastushok L, Ptak C, Xiao W, Ellison MJ. Noncovalent interaction between ubiquitin and the human DNA repair protein Mms2 is required for Ubc13-mediated polyubiquitination. J Biol Chem. 2001;276(43):40120–6.View ArticlePubMedGoogle Scholar
- Hofmann RM, Pickart CM. Noncanonical MMS2-encoded ubiquitin-conjugating enzyme functions in assembly of novel polyubiquitin chains for DNA repair. Cell. 1999;96(5):645–53.View ArticlePubMedGoogle Scholar
- Guo H, Wen R, Wang Q, Datla R, Xiao W. Three Brachypodium distachyon Uev1s promote Ubc13-mediated Lys63-linked polyubiquitination and confer different functions. Front Plant Sci. 2016;7:1551.PubMedPubMed CentralGoogle Scholar
- Yalovsky S, Rodr Guez-Concepcion M, Gruissem W. Lipid modifications of proteins - slipping in and out of membranes. Trends Plant Sci. 1999;4(11):439–45.View ArticlePubMedGoogle Scholar
- Wang M, Casey PJ. Protein prenylation: unique fats make their mark on biology. Nat Rev Mol Cell Bio. 2016;17(2):110–22.View ArticleGoogle Scholar
- Pastushok L, Spyracopoulos L, Xiao W. Two Mms2 residues cooperatively interact with ubiquitin and are critical for Lys63 polyubiquitination in vitro and in vivo. FEBS Lett. 2007;581(28):5343–8.View ArticlePubMedGoogle Scholar
- Lewis MJ, Saltibus LF, Hau DD, Xiao W, Spyracopoulos L. Structural basis for non-covalent interaction between ubiquitin and the ubiquitin conjugating enzyme variant human MMS2. J Biomol NMR. 2006;34(2):89–100.View ArticlePubMedGoogle Scholar
- James P, Halladay J, Craig EA. Genomic libraries and a host strain designed for highly efficient two-hybrid selection in yeast. Genetics. 1996;144(4):1425–36.PubMedPubMed CentralGoogle Scholar
- Andersen PL, Zhou HL, Pastushok L, Moraes T, McKenna S, Ziola B, Ellison MJ, Dixit VM, Xiao W. Distinct regulation of Ubc13 functions by the two ubiquitin-conjugating enzyme variants Mms2 and Uev1A. J Cell Biol. 2005;170(5):745–55.View ArticlePubMedPubMed CentralGoogle Scholar
- Yin XJ, Volk S, Ljung K, Mehlmer N, Dolezal K, Ditengou F, Hanano S, Davis SJ, Schmelzer E, Sandberg G, et al. Ubiquitin lysine 63 chain forming ligases regulate apical dominance in Arabidopsis. Plant Cell. 2007;19(6):1898–911.View ArticlePubMedPubMed CentralGoogle Scholar
- Li W, Schmidt W. A lysine-63-linked ubiquitin chain-forming conjugase, UBC13, promotes the developmental responses to iron deficiency in Arabidopsis roots. Plant J. 2010;62(2):330–43.View ArticlePubMedGoogle Scholar
- Mural RV, Liu Y, Rosebrock TR, Brady JJ, Hamera S, Connor RA, Martin GB, Zeng L. The tomato Fni3 lysine-63-specific ubiquitin-conjugating enzyme and suv ubiquitin E2 variant positively regulate plant immunity. Plant Cell. 2013;25(9):3615–31.View ArticlePubMedPubMed CentralGoogle Scholar
- Wen R, Wang S, Xiang D, Venglat P, Shi X, Zang Y, Datla R, Xiao W, Wang H. UBC13, an E2 enzyme for Lys63-linked ubiquitination, functions in root development by affecting auxin signaling and aux/IAA protein stability. Plant J. 2014;80(3):424–36.View ArticlePubMedGoogle Scholar
- Guo H, Wen R, Liu Z, Datla R, Xiao W. Molecular cloning and functional characterization of two Brachypodium distachyon UBC13 genes whose products promote K63-linked polyubiquitination. Front Plant Sci. 2016;6:1222.PubMedPubMed CentralGoogle Scholar
- Fritsche J, Rehli M, Krause SW, Andreesen R, Kreutz M. Molecular cloning of a 1alpha,25-dihydroxyvitamin D3-inducible transcript (DDVit 1) in human blood monocytes. Biochem Biophys Res Commun. 1997;235(2):407–12.View ArticlePubMedGoogle Scholar
- Ma L, Broomfield S, Lavery C, Lin SL, Xiao W, Bacchetti S. Up-regulation of CIR1/CROC1 expression upon cell immortalization and in tumor-derived human cell lines. Oncogene. 1998;17(10):1321–6.View ArticlePubMedGoogle Scholar
- Sancho E, Vila MR, Sanchez-Pulido L, Lozano JJ, Paciucci R, Nadal M, Fox M, Harvey C, Bercovich B, Loukili N, et al. 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. 1998;18(1):576–89.View ArticlePubMedPubMed CentralGoogle Scholar
- Sherman F, Fink GR, Hicks J. Methods in yeast genetics. Cold Spring Harbor, NY: Cold Spring Harbor Laboratory Press; 1983.Google Scholar
- Ito H, Fukuda Y, Murata K, Kimura A. Transformation of intact yeast cells treated with alkali cations. J Bacteriol. 1983;153(1):163–8.PubMedPubMed CentralGoogle Scholar
- Bartel PL, Fields S. Analyzing protein-protein interactions using two-hybrid system. Methods Enzymol. 1995;254:241–63.View ArticlePubMedGoogle Scholar
- Xu X, Lambrecht AD, Xiao W. Yeast survival and growth assays. Methods Mol Biol. 2014;1163:183–91.View ArticlePubMedGoogle Scholar
- Waadt R, Kudla J: In Planta Visualization of Protein Interactions Using Bimolecular Fluorescence Complementation (BiFC). Cold Spring Harbor Protocols 2008, 2008(5):pdb.prot4995-pdb.prot4995.Google Scholar