Systematic characterization of Brassica napus UBC13 genes involved in DNA-damage response and K63-linked polyubiquitination

Abstract

Background: Ubc13 is the only known ubiquitin conjugating enzyme (Ubc/E2) dedicated to promoting Lys (K)63-linked polyubiquitination, and this process requires a Ubc/E2 variant (UEV). Unlike conventional K48-linked polyubiquitination that targets proteins for degradation, K63-linked polyubiquitination, which is involved in several cellular processes, does not target proteins for degradation but alter their activities.

Results: In this study we report the identification and functional characterization of 12 Brassica napus UBC13 genes. All the cloned UBC13 gene products were able to physically interact with AtUev1D, an Arabidopsis UEV, to form stable complexes that are capable of catalyzing K63-linked polyubiquitination in vitro. Furthermore, BnUBC13 genes functionally complemented the yeast ubc13 null mutant defects in spontaneous mutagenesis and DNA-damage responses, suggesting that BnUBC13s can replace yeast UBC13 in mediating K63-linked polyubiquitination and error-free DNA-damage tolerance.

Conclusion: Collectively, this study provides convincing data to support notions that B. napus Ubc13s promote K63-linked polyubiquitination and are probably required for abiotic stress response. Since plant Ubc13-UEV are also implicated in other developmental and stress responses, this systematic study sets a milestone in exploring roles of K63-linked polyubiquitination in this agriculturally important crop.

Keywords: Abiotic response; Brassica napus; DNA-damage tolerance; K63-linked polyubiquitination; Protein-protein interaction.

Fig. 1

Sequence analysis of Ubc13s from B. napus (Bn) and A. thaliana (At). A Predicted amino acid sequences of UBC13 gene products from B. napus and Arabidopsis were aligned by the BioEdit software version 7.2.5. Identical residues shared by the majority of Ubc13s are highlighted. Amino acid residues required for the interaction with UEV are marked by red asterisks, required for the interaction with a RING E3 is marked by a blue asterisk, and the active site Cys residue is marked by a green asterisk. B A phylogenetic tree based on B. napus and Arabidopsis Ubc13 family amino acid sequences was constructed by using MEGA7.0.26 and iTOL. Lines with different colors represent different branches: black, Arabidopsis; red and purple, two separate branches of B. napus. Yellow and green rectangles highlight Ubc13 proteins from B. napus and Arabidopsis, respectively. Purple circles indicate bootstrap levels as indicated

Fig. 2

Functional complementation of the yeast ubc13 null mutant by BnUBC13s. WXY904 (ubc13∆) cells transformed with vector pGBT9E or plasmids carrying indicated BnUBC13 genes were grown in the selective medium overnight and the cell density was adjusted. A A serial dilution assay. Cells were diluted and printed onto YPD plates with or without different concentrations of 4NQO or MMS. For the UV irradiation, plates containing printed cells were exposed to 254 nm UV at given doses. B A gradient plate assay. Cells were printed to a square plate across the MMS gradient at given concentrations. The plates were incubated at 30 ºC for 2 days before being photographed. The arrow points to increasing MMS concentrations. Several doses of DNA-damaging agents were examined and only one representative plate is shown. For each sample, several independent colonies from each transformation were examined with comparable results, and only one set of plates is shown. Wild type, HK578-10D.

Fig. 3

Functional complementation of the yeast ubc13 rev3 double mutant by BnUBC13s. WXY904 (ubc13Δ) and WXY921 (ubc13Δ rev3Δ) cells transformed with vector pGBT9E or plasmids carrying indicated BnUBC13 genes were grown in the selective medium overnight. After adjustment of the cell density, cells were printed onto plates containing different concentrations of MMS gradient, and the plates were incubated at 30 ºC for 2 days before being photographed. The arrow points to increasing MMS concentrations. Several independent colonies from each transformation were examined with comparable results, and only one set of plates is shown here. Wild type, HK578-10D; rev3Δ, WXY1233.

Fig. 4

Effects of BnUBC13s on spontaneous mutagenesis. WXY849 (ubc13∆) cells transformed with vector pGAD424E or plasmids carrying BnUBC13 genes were grown in the selective medium, diluted in YPD, incubated at 30 ^oC for 3 days, plated onto SD-Leu and SD-Leu-Trp, and the plates were incubated for 3 days before counting number of colonies. Spontaneous mutation rates were calculated [54] and presented as mutation events per cell per generation. Wild type, DBY747

Fig. 5

Physical interactions between BnUbc13s and AtUev1D in a yeast two-hybrid assay. PJ69-4 A cells co-transformed with vectors pGBT9E (Gal4_BD) and pGAD424E (Gal4_AD) or plasmids carrying AtUEV1D and indicated BnUBC13s were grown in the SD-Leu-Trp medium overnight. After adjustment of cell density, cells were replicated on SD-Trp-Leu (control), SD-Trp-Leu-His plus various concentrations of 3-AT, and SD-Trp-Leu-Ade, followed by incubation for 3 days at 30 ^oC before being photographed. Five independent colonies from each transformation were examined with comparable results, and only one set is shown here

Fig. 6

Physical interactions between BnUbc13s and AtUev1D in a GST pulldown assay. Co-purified GST-AtUev1D and His₆-BnUbc13s were added to microspin columns. After incubation and washing, the columns were eluted with reduced glutathione and subjected to SDS-PAGE analysis. A AtUev1D with BnUbc13A-D. B AtUev1D with BnUbc13E-H. Identities of key bands are marked. Unprocessed WB images shown in A, B are given in Supplementary Fig. S3A, B

Fig. 7

Functional complementation of the yeast ubc13 mms2 double mutant by BnUBC13s and AtUEV1D. WXY955 (ubc13Δ mms2Δ) cells co-transformed with vectors pGBT9E and pGAD424E or with plasmids containing AtUEV1D and indicated BnUBC13s were grown in the SD-Leu-Trp selective medium overnight. After adjustment with cell density, cells were printed onto YPD plates containing 0.025% MMS gradient and the plates were incubated at 30 ºC for 2 days before being photographed. The arrow points to increasing MMS concentrations. Several independent colonies from each transformation were examined with comparable results, and only one set of plates is shown here. Wild type, HK578-10D.

Fig. 8

The in vitro Ub conjugation assay using purified BnUbc13s and AtUev1D. A Ub conjugation by BnUbc13A, B, C and AtUev1D. B Ub conjugation by BnUbc13D, E and AtUev1D. An in vitro Ub conjugation assay was performed using purified proteins as indicated. Assay samples were subjected to SDS-PAGE and western blotting analyses using an anti-Ub antibody. Free Ub and poly-Ub chains are marked. Unprocessed WB images shown in A, B are given in Supplementary Fig. S4A, B