Rice UBC13, a candidate housekeeping gene, is required for K63-linked polyubiquitination and tolerance to DNA damage
© Zang et al.; licensee Springer. 2012
Received: 13 March 2012
Accepted: 26 July 2012
Published: 8 September 2012
While plant growth and reproduction is dependent on sunlight, UV irradiation from sunlight is one of the major genotoxic stresses that threaten plant survival and genome stability. In addition, many environmental chemicals can also damage the plant genome. In yeast and mammalian cells protection against the above genome instability is provided by an error-free DNA-damage tolerance (DDT) pathway, which is dependent on Ubc13-mediated K63-linked polyubiquitination of the proliferating cell nuclear antigen (PCNA). In this study, we isolated the UBC13 gene from rice and characterized its functions. Expression of OsUBC13 can protect a yeast ubc13 null mutant against spontaneous and environmental DNA damage. Furthermore, OsUbc13 physically interacts with human Ubc13 partners Mms2 and Uev1A, and catalyzes K63 polyubiquitination in vitro. These observations collectively suggest that the K63 polyubiquitination is conserved in rice, and that OsUBC13 may be involved in DDT and other cellular processes. In addition, OsUBC13 is constitutively expressed at a high level even under various stress conditions, suggesting that it is a housekeeping gene.
Ubiquitination is an essential protein posttranslational modification (PTM) process found in all eukaryotic organisms. This modification by a 76 amino acid ubiquitin (Ub) determines protein turnover, regulation and molecular function, and provides a complex regulation of various biological processes ([Hershko and Ciechanover 1998]). The most well-understood ubiquitination pathways are the ubiquitin-proteasome system (UPS) that mediates protein degradation ([Hochstrasser 1996]). UPS has been reported to be involved in almost all aspects of plant biology, including phytohormone signalling (auxin, gibberellins, jasmonic acid etc.), plant morphogenesis, immune response, self-incompatibility, chromatin modification and epigenetic regulation ([Vierstra 2009]). Ubiquitination, as an enzymatic cascade, is catalyzed by members of three enzyme families: ubiquitin-activating enzyme (Uba or E1), ubiquitin-conjugating enzyme (Ubc or E2) and ubiquitin ligase (Ubl or E3) enzymes. Before conjugation takes place, the initial activation of ubiquitin is performed by E1 in an ATP-dependent manner. The activated ubiquitin is then transferred from E1 to an active-site cysteine of E2 via a trans-esterification reaction. Finally, a Ub-loaded E2 selectively interacts with a cognate E3 that recruits a specific substrate. The C-terminal glycine of Ub is linked to the ϵ-amino group lysine residue of either the target protein or a previously attached Ub ([Hershko and Ciechanover 1998]; van [Wijk and Timmers 2009]). Unlike HECT (homologous to E6-AP C terminus) E3s that first accept the activated Ub prior to substrate modification ([Rotin and Kumar 2009]), RING (really interesting new gene) and U-box E3s act as a platform and provide an optimal conformation for the Ub transfer while binding both the substrate and Ub-loaded E2 ([Ardley and Robinson 2005]; [Deshaies and Joazeiro 2009]).
In this enzymatic cascade, the highly-conserved E2 family that couples the activation of Ub to the conjugation event appears to play a central role. E2s directly influence which surface lysine residue in Ub will be attached to the incoming Ub, which affects the destiny of the substrate (Burroughs et al. ; [Pickart 2000]; [Pickart and Fushman 2004]). In the UPS pathway, the target protein is modified by a Lys48 (K48)-linked poly-Ub chain that is recognized and degraded by 26 S proteasome ([Hershko and Ciechanover 1998]). In Arabidopsis this process is catalyzed by at least 37 E2s and accompanied by potentially over 1,400 different E3s to recognize different substrates in various biological pathways (Kraft et al. ; [Vierstra 2009]). In contrast, only one E2, Ubc13, has been reported to be capable of catalyzing K63-linked poly-Ub chain assembly in eukaryotes, from unicellular yeasts to human, and this non-canonical poly-Ub chain assembly requires a heterodimeric E2 complex consisting of Ubc13 and a Ubc-E2 variant (Uev) ([Hofmann and Pickart 1999]; McKenna et al. ). In Saccharomyces cerevisiae, Ubc13-Uev (Mms2) complex coupled with the E3 Rad5 modifies proliferating cell nuclear antigen (PCNA) at its K164 residue with K63-linked poly-Ub chain, which promotes error-free DNA-damage tolerance (DDT, also known as post-replication repair, PRR) as one of two means of bypassing replication-blocking lesions (Broomfield et al. ; Hoege et al. ; [Hofmann and Pickart 1999]; [Ulrich and Jentsch 2000]). Another lesion bypass method is called translesion DNA synthesis (TLS), which utilizes non-essential DNA polymerases including Polη (Rev3 + Rev7), Polζ, and Rev1 ([Ulrich and Jentsch 2000]; Xiao et al. ). It is now clear that the K164 residue of PCNA is monoubiquitinated by the E2-E3 complex Rad6-Rad18, which promotes TLS, whereas its sequential K63-linked polyubiquitination by Mms2-Ubc13-Rad5 promotes error-free DDT (Hoege et al. ; [Pastushok and Xiao 2004]). Interestingly, the PCNA-K164 residue can also be SUMOylated by the E2-E3 complex Siz1-Ubc9 (Hoege et al. ; [Stelter and Ulrich 2003]). Consequently, the SUMOylated PCNA can recruit the DNA helicase Srs2 to stalled replication forks to prevent unwanted homologous recombination (Papouli et al. ; Pfander et al. ).
Plants, as sessile lifestyle organisms, have to face numerous environmental stresses including genotoxic stress, such as UV irradiation and chemical pollutants. This wide range of stresses threatens plants survival, reduces crop yields and food quality, and compromises world food security. Although there are some reports on genes involved in plant DDT, most describe TLS polymerases (Anderson et al. ; [Curtis and Hays 2007]; Garcia-Ortiz et al. ; Sakamoto et al. ; Takahashi et al. ) and a few on error-free DDT genes from Arabidopsis (Chen et al. ; Wen et al. ; Wen et al. ), with little elucidation on the mechanism. Surprisingly, to the best of our knowledge, there is no report on the isolation and characterization of genes involved in DDT in crop plants. In this study, the rice UBC13 gene was isolated and functionally characterized. It was found that OsUBC13 is able to functionally complement the yeast ubc13 null mutant and that OsUbc13 protein can interact with Uevs from other species and mediate K63-linked polyubiquitination in vitro, suggesting that it may play similar roles in rice. It was also found that both the transcript and protein levels of OsUbc13 remain stable during plant growth and development, as well as facing various biotic and abiotic stresses. We argue that OsUBC13 is a candidate housekeeping gene and its expression may be used as a useful internal control for the study of other rice genes or gene products.
Identification and sequence analysis of the OsUBC13 gene
OsUBC13 Functionally complements yeast ubc13 null mutant
Spontaneous mutation rates
1.28 ± 0.30
31.43 ± 9.48
6.76 ± 0.33
OsUbc13 Physically interacts with yeast and human E2 variants
To further confirm the yeast two-hybrid results, a glutathione S-transferase (GST)-affinity pull-down assay was performed (Figure 3c). In E. coli cells co-transformed with plasmids expressing His6-OsUbc13 and GST-Uev, His6-OsUbc13 could be co-purified by GST-hUev1A (Lane 2) and GST-hMms2 (Lane 4) from total cell lysates. As a negative control, GST alone (Lane 6) was unable to pull-down His6-OsUbc13 under the same experimental conditions. Furthermore, previous yeast complementation experiments indicate that heterologous Ubc13 must physically interact with the endogenous yMms2, which is the prerequisite for the functional complementation (Pastushok et al. ). Taken together, these data collectively suggest that all three Uevs from human and yeast can form stable heterodimers with OsUbc13 in vitro and in vivo.
OsUbc13 Mediates K63-linked polyubiquitination with human Uevs in vitro
OsUbc13 Is ubiquitously expressed and does not fluctuate under biotic or abiotic stresses
Although the transcript level of OsUBC13 is relatively stable, it does not rule out the possibility that OsUBC13 may be regulated at the protein level. To further determine the cellular Ubc13 protein level under various stresses, we used the monoclonal antibody 4E11 produced in-house against hUbc13 (Andersen et al. ), which shares a very high degree of amino acid sequence identity with OsUbc13 (Figure 1a). A single band of approximately 20 kDa was detected from total rice seedling extract in a western blot analysis, consistent with the predicted OsUbc13 protein size. In this study, abscisic acid (ABA), salicylic acid (SA) and jasmonic acid (JA) were selected to represent hormone treatments, while methyl methanesulfonate (MMS), cisplatin and hydrogen peroxide (H2O2) were chosen to represent DNA damage treatments. As shown in Figure 5b and quantitated by densitometry (data not shown), no obvious alteration was observed by any given treatments under our experimental conditions.
While sunlight is absolutely necessary for photosynthesis by land plants, the threats of solar UV radiation as well as other genotoxic chemicals are also critical to plants survival due to their sessile living style. As the major food resources for human beings, crops may severely lose yield and quality due to the above stresses, which compromise global food security. In this study, we isolated and characterized the rice UBC13 gene, which is believed to be the first reported crop gene involved in the DNA damage-tolerance pathway. Our in vitro studies confirmed that the highly conserved OsUbc13 protein could physically interact with the cognate E2 variants from yeast and human. Functional examination revealed that OsUBC13 could restore cellular activities of the yeast ubc13 null mutant to limit spontaneous mutagenesis and provide resistance to DNA-damaging agents. Since yeast UBC13 plays an essential role in mediating error-free DDT (Broomfield et al. ), these results suggest that OsUBC13 is involved in the error-free DDT pathway in rice as well, although definitive evidence has to wait for the characterization of UBC13 knockdown or knockout rice plants. It is noticed that rice may be a more suitable experimental plant model than other model plants including Arabidopsis, since it contains only one predicted UBC13 gene, while Arabidopsis contains two highly conserved and most likely redundant UBC13 genes (Wen et al. ). Furthermore, since the DDT pathway appears to be conserved from yeast to humans (Andersen et al. ), this study may serve as a gateway to understanding DDT mechanisms in rice in an attempt to enhance its resistance to this major environment stress.
Ubc13 is the only known E2 that catalyzes K63-linked polyubiquitination. This catalytic activity is dependent on its interaction with an E2 variant (Uev) and Ubc13 associated with different Uevs is thought to mediate different cellular processes (Andersen et al. ). In S. cerevisiae, Ubc13 has only one Uev partner, Mms2, and the Ubc13-Mms2 heterodimer is required for the K63 polyubiquitination of PCNA (Hoege et al. ). In human cells, two Ubc13-associated Uevs are found (Xiao et al. ), and they appear to play different roles (Andersen et al. ): the Ubc13-hMms2 complex is required for DNA-damage response, while the Ubc13-Uev1A complex is required for the NF-κB signaling pathway (Deng et al. ) through activation of NEMO (Zhou et al. ) and/or TAK1 (Wang et al. ). In this study, we found that OsUbc13 forms stable complexes with both hMms2 and Uev1A, and could catalyze K63-linked poly-Ub chains in vitro with either Uev1A or hMms2. Interestingly, Arabidopsis contains four UEV1 genes; at least one of them (UEV1D) is involved in DNA-damage response, while at least one (UEV1A) is not required for this response (Wen et al. ). Since the rice genome also contains multiple UEV1 homologs (Y.Z. and W.X., data not shown), it is conceivable that rice Ubc13 associates with different Uev1s to catalyze K63-linked poly-Ub chains. These different heterodimers may interact with different E3s, target proteins and hence are involved in various stress responses including, but not limited to, DNA-damage tolerance. Indeed, Ubc13-mediated ubiquitination has been reported to be involved in cellular processes such as neurodegeneration (Doss-Pepe et al. ; Lim et al. ; [Muralidhar and Thomas 1993]; Oh et al. ), homologous recombination (Doil et al. ; Huen et al. ; Mailand et al. ; Stewart et al. ), innate immunity (Deng et al. ; Zhou et al. ), cell cycle checkpoint (Bothos et al. ; Laine et al. ; Wen et al. ), as well as development (Yin et al. ) and iron metabolism ([Li and Schmidt 2010]) in Arabidopsis. Some of these processes may not necessarily require a Uev (Huen et al. ) or involve K63-linked polyubiquitination.
K63-linked polyubiquitination process is primarily involved in stress responses, within which the E2 complex plays a critical role. It is reasonable to assume that the Ubc13-Uev activity is tightly regulated in response to stress. In S. cerevisiae, UBC13 is transcriptionally regulated in response to DNA damage. In higher eukaryotes, because Ubc13 is involved in multiple cellular processes, and whose specificity is largely determined by the cognate Uev, regulation of Uev activity would be evolutionarily preferred. Indeed, there is little experimental evidence on altered Ubc13 activity in mammalian cells, while cellular Uev levels appear to fluctuate in different tissues and in response to environmental stresses (Fritsche et al. ; Ma et al. ; Sancho et al. ). Of great interest is the observation that transcript levels of UBC13 genes in Arabidopsis and rice are relatively stable regardless of plant development, tissue distribution and response to stresses, indicating that UBC13 is a housekeeping gene in plants. In sharp contrast, the expression of plant UEV1 genes fluctuate under the same experimental conditions, suggesting that plants achieve the control of K63 polyubiquitination through differential regulation of UEV1 genes, or that Uevs serve as a regulatory subunit for K63 ubiquitination. This molecular evolution further testifies that in higher eukaryotes, K63 ubiquitination is involved in multiple cellular processes.
Since transcriptional regulation is only one of several means of controlling target gene activity, in this study we attempted to assess the cellular Ubc13 protein level under different stress conditions. Western blot analysis by using an anti-hUbc13 monoclonal antibody further confirms that UBC13 is a candidate housekeeping gene. Hence, we recommend Ubc13 to be a suitable candidate to serve as an internal reference for rice-related research because it fulfills the following criteria: (a) it is a highly expressed gene; (b) its expression level is remarkably constant under various growth stages, tissues and experimental conditions; (c) there is only one copy of the homologous gene; and (d) the monoclonal antibody 4E11 used in this study is commercially available.
The present study describes the isolation and functional characterization of the rice UBC13 (OsUBC13) gene and its product. Our observations collectively suggest that Ubc13-mediated K63 polyubiquitination is conserved in rice, and that OsUBC13 may be involved in DNA damage tolerance as well as other cellular stress responses. Furthermore, bioinformatic analysis and our experimental data suggest that OsUBC13 is a housekeeping gene, and that its expression can be utilized as an internal control for other rice-related investigations.
Plant materials and yeast cell culture
Rice (Oryza sativa L. cv. Japonaca) seeds were surface sterilized with 2% NaClO for 30 min after pre-wash by sterilized distilled water, followed by washing seven times in sterilized water. The sterilized rice seeds were plated in ½ Murashige and Skoog (MS) plates containing MS with 2.2 g/l minimal organics (Sigma M6899), 10 g/l sucrose and 1% agar (BD 214010), and vertically cultured in a growth chamber (16 h light/8 h dark and 30°C constant). Two weeks after seed germination, rice seedlings were transferred to ½ MS solution for an additional 1-day incubation. For ABA (final concentration 50 μM), MMS (final concentration of 0.175%) and cisplatin (final concentration 16 μM) treatments, stock solutions were added to the nutrient solution to the predetermined final concentration. For SA and meJA treatments, SA (5 mM in water) and meJA (0.1 mM in 0.1% ethanol) were sprayed onto the rice shoot. For ABA, SA, meJA and H2O2 treatments, the seedlings were harvested after 8 hours. For MMS and cisplatin treatments, seedlings were harvested after 12 hours.
The haploid yeast strains used in this study are listed in Additional file 4: Table S1. 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. ). For solid plates, 2% agar was added to either YPD or SD medium prior to autoclaving. Yeast cells were transformed using a LiAc method as described (Ito et al. ). The source and preparation of ubc13Δ::HIS3 cassettes was as previously described (Xiao et al. ).
Cloning rice UBC13 cDNA and plasmid construction
To clone the full-length open reading frame (ORF) of OsUBC13, total RNA was isolated from rice seedlings by using TRIzol reagents (Invitrogen, Carlsbad, CA, USA) for RT-PCR with the RevertAid First Strand cDNA Synthesis Kit (Fermentas) following the manufacturer’s protocols. OsUbc13 specific primers are as follow: 5’-aatccggaattc ATGGCCAACAGCAACCTC-3’ and 5’-atacgcgtcgac TTATGCACCGCTGGC-3’. The forward primer contains an Eco RI restriction site and the reverse primer contains a Sal I site, as underlined. The PCR product of OsUBC13 ORF was cloned into yeast two-hybrid vectors pGBT9Bg and pGAD424Bg, which were derived from pGBT9 and pGAD424 ([Bartel and Fields 1995]), respectively.
Yeast two-hybrid analysis
Recombinant protein purification and ubiquitination assay
The OsUbc13 ORF was isolated from pGBT-OsUbc13 and cloned into pET-30a. The resulting pET-OsUbc13 was transformed into BL21 CodonPlus (DE3)-RIL cells. The His6-OsUbc13 fusion protein was purified following a previously published protocol (Anderson et al. ). Meanwhile, GST, GST-hUev1A and GST-hMms2 were produced and purified as previously described (Andersen et al. ). For an in vitro ubiquitination assay, the 20-μl reaction mixture contained 225 nM E1 enzyme, 450 μM Ub, 1 mM Mg-ATP, 1 μM Ubc13, and 1 μM Uev1A in the supplied reaction buffer (Boston Biochem). The K63R and K48R mutant Ub proteins were also purchased from Boston Biochem. The conjugation reactions were performed at 37°C for 2 hr. Samples were subjected to SDS-PAGE (12%), and Ub and poly-Ub were detected through protein gel blots using polyclonal rabbit anti-Ub antibodies (Sigma-Aldrich).
GST pull-down assay
The BL21 competent cells were transformed with either pGEX6p-1, pGEX-hMms2 or pGEX-hUev1A alone, or co-transformed with pET-OsUbc13. The whole cell extracts were incubated with Glutathione Sepharose 4B MicrospinTM beads in 4°C for 2 hr. The beads were collected by centrifugation and washed with a lysis buffer 5 times. Finally, beads were boiled with loading buffer and analyzed on a 12% SDS-PAGE gel.
Rice protein extraction and western blot analysis
Two-week-old rice seedlings were homogenized in liquid nitrogen. Total protein was extracted using a buffer containing 50 mM Tris–HCl pH8.0, 0.3 M NaCl, 2 mM EDTA, 10% Glycerol, 0.1% TritonX-100, 10 mM PMSF, 3 mM DDT and 1/5 tablet per 10 ml protease inhibitor (Roche). The extract was centrifuged at 16,000 g for 10 min at 4°C. The supernatant was then transferred to a new tube and centrifuged twice prior to SDS/PAGE analysis. The OsUbc13 protein in each sample was detected by western bolt using the anti-hUbc13 monoclonal antibody 4E11 (Andersen et al. ).
Yeast cell survival assays
Yeast strain HK578-10D and its isogenic ubc13Δ single mutant transformed with pGAD424 or pGAD-OsUbc13, were used in this assay. The yeast liquid killing and serial dilution assays were performed as previously described (Barbour et al. ; Wang et al. ). For the serial dilution assay, several doses for each DNA-damaging agent were employed and only one representative dose is presented.
Spontaneous mutagenesis assay
Yeast strain DBY747 and its isogenic ubc13Δ derivative WXY849 bear a trp1-289 amber mutation that can be reverted to Trp+ by several different mutation events ([Xiao and Samson 1993]). WXY849 was transformed with pGAD-OsUbc13 and the spontaneous mutagenesis assay was performed as described previously (Anderson et al. ).
The authors wish to thank Guang Yang, Xin Xu and Amanda Lambrecht for technical assistance and other Xiao laboratory members for helpful discussion. This work was supported by an operating grant (No. KZ201110028037) from Beijing Education Committee.
- Andersen PL, Zhou H, 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: 745–755. 10.1083/jcb.200502113PubMed CentralView ArticlePubMedGoogle Scholar
- Anderson HJ, Vonarx EJ, Pastushok L, Nakagawa M, Katafuchi A, Gruz P, Di Rubbo A, Grice DM, Osmond MJ, Sakamoto AN, Nohmi T, Xiao W, Kunz BA: Arabidopsis thaliana Y-family DNA polymerase η catalyses translesion synthesis and interacts functionally with PCNA2. Plant J 2008, 55: 895–908. 10.1111/j.1365-313X.2008.03562.xView ArticlePubMedGoogle Scholar
- Ardley HC, Robinson PA: E3 ubiquitin ligases. Essays Biochem 2005, 41: 15–30. 10.1042/EB0410015View ArticlePubMedGoogle Scholar
- Barbour L, Ball LG, Zhang K, Xiao W: DNA damage checkpoints are involved in postreplication repair. Genetics 2006, 174: 1789–1800. 10.1534/genetics.106.056283PubMed CentralView ArticlePubMedGoogle Scholar
- Bartel PL, Fields S: Analyzing protein-protein interactions using two-hybrid system. Methods Enzymol 1995, 254: 241–263.View ArticlePubMedGoogle Scholar
- Bothos J, Summers MK, Venere M, Scolnick DM, Halazonetis TD: The Chfr mitotic checkpoint protein functions with Ubc13-Mms2 to form Lys63-linked polyubiquitin chains. Oncogene 2003, 22: 7101–7107. 10.1038/sj.onc.1206831View 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 USA 1998, 95: 5678–5683. 10.1073/pnas.95.10.5678PubMed CentralView ArticlePubMedGoogle Scholar
- Broomfield S, Hryciw T, Xiao W: DNA postreplication repair and mutagenesis in Saccharomyces cerevisiae . Mutat Res 2001, 486: 167–184. 10.1016/S0921-8777(01)00091-XView ArticlePubMedGoogle 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: 168–174. 10.1007/s002940050515View ArticlePubMedGoogle Scholar
- Burroughs AM, Jaffee M, Iyer LM, Aravind L: Anatomy of the E2 ligase fold: implications for enzymology and evolution of ubiquitin/Ub-like protein conjugation. J Struct Biol 2008, 162: 205–218. 10.1016/j.jsb.2007.12.006PubMed CentralView ArticlePubMedGoogle Scholar
- Chen IP, Mannuss A, Orel N, Heitzeberg F, Puchta H: A homolog of ScRAD5 is involved in DNA repair and homologous recombination in Arabidopsis. Plant Physiol 2008, 146: 1786–1796. 10.1104/pp.108.116806PubMed CentralView ArticlePubMedGoogle Scholar
- Curtis MJ, Hays JB: Tolerance of dividing cells to replication stress in UVB-irradiated Arabidopsis roots: requirements for DNA translesion polymerases η and ζ. DNA Repair. 2007, 6: 1341–1358. 10.1016/j.dnarep.2007.03.004View ArticlePubMedGoogle Scholar
- Deng L, Wang C, Spencer E, Yang L, Braun A, You J, Slaughter C, Pickart C, Chen ZJ: Activation of the IκB kinase complex by TRAF6 requires a dimeric ubiquitin-conjugating enzyme complex and a unique polyubiquitin chain. Cell 2000, 103: 351–361. 10.1016/S0092-8674(00)00126-4View ArticlePubMedGoogle Scholar
- Deshaies RJ, Joazeiro CA: RING domain E3 ubiquitin ligases. Annu Rev Biochem 2009, 78: 399–434. 10.1146/annurev.biochem.78.101807.093809View ArticlePubMedGoogle Scholar
- Doil C, Mailand N, Bekker-Jensen S, Menard P, Larsen DH, Pepperkok R, Ellenberg J, Panier S, Durocher D, Bartek J, Lukas J, Lukas C: RNF168 binds and amplifies ubiquitin conjugates on damaged chromosomes to allow accumulation of repair proteins. Cell 2009, 136: 435–446. 10.1016/j.cell.2008.12.041View ArticlePubMedGoogle Scholar
- Doss-Pepe EW, Chen L, Madura K: α-Synuclein and Parkin contribute to the assembly of ubiquitin lysine 63-linked multiubiquitin chains. J Biol Chem 2005, 280: 16619–16624. 10.1074/jbc.M413591200View ArticlePubMedGoogle Scholar
- Fields S, Song O: A novel genetic system to detect protein-protein interactions. Nature 1989, 340: 245–246. 10.1038/340245a0View ArticlePubMedGoogle Scholar
- Fritsche J, Rehli M, Krause SW, Andreesen R, Kreutz M: Molecular cloning of a 1α,25-dihydroxyvitamin D3-inducible transcript (DDVit 1) in human blood monocytes. Biochem Biophys Res Commun 1997, 235: 407–412. 10.1006/bbrc.1997.6798View 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: 84–97. 10.1111/j.1365-313X.2004.02112.xView ArticlePubMedGoogle Scholar
- Hershko A, Ciechanover A: The ubiquitin system. Annu Rev Biochem 1998, 67: 425–479. 10.1146/annurev.biochem.67.1.425View ArticlePubMedGoogle Scholar
- Hochstrasser M: Ubiquitin-dependent protein degradation. Annu Rev Genet. 1996, 30: 405–439.PubMedGoogle 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: 135–141. 10.1038/nature00991View 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: 645–653. 10.1016/S0092-8674(00)80575-9View ArticlePubMedGoogle Scholar
- Hofmann RM, Pickart CM: In vitro assembly and recognition of Lys-63 polyubiquitin chains. J Biol Chem 2001, 276: 27936–27943. 10.1074/jbc.M103378200View ArticlePubMedGoogle Scholar
- Hruz T, Laule O, Szabo G, Wessendorp F, Bleuler S, Oertle L, Widmayer P, Gruissem W, Zimmermann P: Genevestigator v3: a reference expression database for the meta-analysis of transcriptomes. Advances in bioinformatics. 2008, 2008: 420747.PubMed CentralView ArticlePubMedGoogle Scholar
- Huen MS, Grant R, Manke I, Minn K, Yu X, Yaffe MB, Chen J: RNF8 transduces the DNA-damage signal via histone ubiquitylation and checkpoint protein assembly. Cell 2007, 131: 901–914. 10.1016/j.cell.2007.09.041PubMed CentralView ArticlePubMedGoogle Scholar
- Huen MS, Huang J, Yuan J, Yamamoto M, Akira S, Ashley C, Xiao W, Chen J: Noncanonical E2 variant-independent function of UBC13 in promoting checkpoint protein assembly. Mol Cell Biol 2008, 28: 6104–6112. 10.1128/MCB.00987-08PubMed CentralView ArticlePubMedGoogle Scholar
- Ito H, Fukuda Y, Murata K, Kimura A: Transformation of intact yeast cells treated with alkali cations. J Bacteriol 1983, 153: 163–168.PubMed CentralPubMedGoogle 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: 1425–1436.PubMed CentralPubMedGoogle Scholar
- Kraft E, Stone SL, Ma L, Su N, Gao Y, Lau OS, Deng XW, Callis J: Genome analysis and functional characterization of the E2 and RING-type E3 ligase ubiquitination enzymes of Arabidopsis. Plant Physiol 2005, 139: 1597–1611. 10.1104/pp.105.067983PubMed CentralView ArticlePubMedGoogle Scholar
- Laine A, Topisirovic I, Zhai D, Reed JC, Borden KL, Ronai Z: Regulation of p53 localization and activity by Ubc13. Mol Cell Biol 2006, 26: 8901–8913. 10.1128/MCB.01156-06PubMed CentralView ArticlePubMedGoogle 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: 330–343. 10.1111/j.1365-313X.2010.04150.xView ArticlePubMedGoogle Scholar
- Lim KL, Chew KC, Tan JM, Wang C, Chung KK, Zhang Y, Tanaka Y, Smith W, Engelender S, Ross CA, Dawson VL, Dawson TM: Parkin mediates nonclassical, proteasomal-independent ubiquitination of synphilin-1: implications for Lewy body formation. J Neurosci 2005, 25: 2002–2009. 10.1523/JNEUROSCI.4474-04.2005View 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: 1321–1326. 10.1038/sj.onc.1202058View ArticlePubMedGoogle Scholar
- Mailand N, Bekker-Jensen S, Faustrup H, Melander F, Bartek J, Lukas C, Lukas J: RNF8 ubiquitylates histones at DNA double-strand breaks and promotes assembly of repair proteins. Cell 2007, 131: 887–900. 10.1016/j.cell.2007.09.040View 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: 40120–40126. 10.1074/jbc.M102858200View ArticlePubMedGoogle Scholar
- Muralidhar MG, Thomas JB: The Drosophila bendless gene encodes a neural protein related to ubiquitin-conjugating enzymes. Neuron 1993, 11: 253–266. 10.1016/0896-6273(93)90182-QView ArticlePubMedGoogle Scholar
- Oh CE, McMahon R, Benzer S, Tanouye MA: bendless , a Drosophila gene affecting neuronal connectivity, encodes a ubiquitin-conjugating enzyme homolog. J Neurosci 1994, 14: 3166–3179.PubMedGoogle Scholar
- Papouli E, Chen S, Davies AA, Huttner D, Krejci L, Sung P, Ulrich HD: Crosstalk between SUMO and ubiquitin on PCNA is mediated by recruitment of the helicase Srs2p. Mol Cell. 2005, 19: 123–133. 10.1016/j.molcel.2005.06.001View 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: 17891–17900.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
- Pfander B, Moldovan GL, Sacher M, Hoege C, Jentsch S: SUMO-modified PCNA recruits Srs2 to prevent recombination during S phase. Nature 2005, 436: 428–433.PubMedGoogle Scholar
- Pickart CM: Ubiquitin in chains. Trends Biochem Sci. 2000, 25: 544–548. 10.1016/S0968-0004(00)01681-9View ArticlePubMedGoogle Scholar
- Pickart CM, Fushman D: Polyubiquitin chains: polymeric protein signals. Curr Opin Chem Biol 2004, 8: 610–616. 10.1016/j.cbpa.2004.09.009View ArticlePubMedGoogle Scholar
- Rotin D, Kumar S: Physiological functions of the HECT family of ubiquitin ligases. Nat Rev Mol Cell Biol 2009, 10: 398–409. 10.1038/nrm2690View 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: 2042–2057. 10.1105/tpc.012369PubMed CentralView ArticlePubMedGoogle Scholar
- Sanada T, Kim M, Mimuro H, Suzuki M, Ogawa M, Oyama A, Ashida H, Kobayashi T, Koyama T, Nagai S, Shibata Y, Gohda J, Inoue J, Mizushima T, Sasakawa C: The Shigella flexneri effector OspI deamidates UBC13 to dampen the inflammatory response. Nature 2012, 483: 623–626. 10.1038/nature10894View ArticlePubMedGoogle Scholar
- Sancho E, Vila MR, Sanchez-Pulido L, Lozano JJ, Paciucci R, Nadal M, Fox M, Harvey C, Bercovich B, Loukili N, Ciechanover A, Lin SL, Sanz F, Estivill X, Valencia A, Thomson TM: 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: 576–589.PubMed CentralPubMedGoogle Scholar
- Sherman F, Fink GR, Hicks J: Methods in Yeast Genetics. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY; 1983.Google Scholar
- Stelter P, Ulrich HD: Control of spontaneous and damage-induced mutagenesis by SUMO and ubiquitin conjugation. Nature 2003, 425: 188–191. 10.1038/nature01965View ArticlePubMedGoogle Scholar
- Stewart GS, Panier S, Townsend K, Al-Hakim AK, Kolas NK, Miller ES, Nakada S, Ylanko J, Olivarius S, Mendez M, Oldreive C, Wildenhain J, Tagliaferro A, Pelletier L, Taubenheim N, Durandy A, Byrd PJ, Stankovic T, Taylor AM, Durocher D: The RIDDLE syndrome protein mediates a ubiquitin-dependent signaling cascade at sites of DNA damage. Cell 2009, 136: 420–434. 10.1016/j.cell.2008.12.042View ArticlePubMedGoogle 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: 870–881. 10.1104/pp.105.060236PubMed CentralView ArticlePubMedGoogle Scholar
- Ulrich HD, Jentsch S: Two RING finger proteins mediate cooperation between ubiquitin-conjugating enzymes in DNA repair. EMBO J 2000, 19: 3388–3397. 10.1093/emboj/19.13.3388PubMed CentralView ArticlePubMedGoogle Scholar
- van Wijk SJL, Timmers HTM: The family of ubiquitin-conjugating enzymes (E2s): deciding between life and death of proteins. FASEB J 2009, 24: 981–993.View ArticlePubMedGoogle Scholar
- VanDemark AP, Hofmann RM, Tsui C, Pickart CM, Wolberger C: Molecular insights into polyubiquitin chain assembly: crystal structure of the Mms2/Ubc13 heterodimer. Cell 2001, 105: 711–720. 10.1016/S0092-8674(01)00387-7View ArticlePubMedGoogle Scholar
- Vierstra RD: The ubiquitin–26 S proteasome system at the nexus of plant biology. Nat Rev Mol Cell Biol 2009, 10: 385–397. 10.1038/nrm2688View ArticlePubMedGoogle Scholar
- Wang C, Deng L, Hong M, Akkaraju GR, Inoue J, Chen ZJ: TAK1 is a ubiquitin-dependent kinase of MKK and IKK. Nature 2001, 412: 346–351. 10.1038/35085597View ArticlePubMedGoogle 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: 620–628. 10.1016/j.dnarep.2011.04.009View ArticlePubMedGoogle Scholar
- Wen R, Li J, Xu X, Cui Z, Xiao W: Zebrafish Mms2 promotes K63-linked polyubiquitination and is involved in p53-mediated DNA-damage response. DNA repair. 2012, 11: 157–166. 10.1016/j.dnarep.2011.10.015View ArticlePubMedGoogle 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: 241–253. 10.1007/s11103-006-0007-xView 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: 213–227. 10.1105/tpc.107.051862PubMed CentralView ArticlePubMedGoogle Scholar
- Wooff J, Pastushok L, Hanna M, Fu Y, Xiao W: The TRAF6 RING finger domain mediates physical interaction with Ubc13. FEBS Lett 2004, 566: 229–233. 10.1016/j.febslet.2004.04.038View 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: 1633–1641.PubMed CentralPubMedGoogle Scholar
- Xiao W, Lin SL, Broomfield S, Chow BL, Wei YF: 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 1998, 26: 3908–3914. 10.1093/nar/26.17.3908PubMed CentralView ArticlePubMedGoogle Scholar
- Xiao W, Samson L: In vivo evidence for endogenous DNA alkylation damage as a source of spontaneous mutation in eukaryotic cells. Proc Natl Acad Sci USA 1993, 90: 2117–2121. 10.1073/pnas.90.6.2117PubMed CentralView ArticlePubMedGoogle Scholar
- Yin XJ, Volk S, Ljung K, Mehlmer N, Dolezal K, Ditengou F, Hanano S, Davis SJ, Schmelzer E, Sandberg G, Teige M, Palme K, Pickart C, Bachmair A: Ubiquitin lysine 63 chain forming ligases regulate apical dominance in Arabidopsis. Plant Cell. 2007, 19: 1898–1911. 10.1105/tpc.107.052035PubMed CentralView ArticlePubMedGoogle Scholar
- Zhou H, Wertz I, O'Rourke K, Ultsch M, Seshagiri S, Eby M, Xiao W, Dixit VM: Bcl10 activates the NF-κB pathway through ubiquitination of NEMO. Nature 2004, 427: 167–171. 10.1038/nature02273View ArticlePubMedGoogle Scholar
This article is published under license to BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.