Os01g0673600

OsUBC13 can protect from spontaneous and environmental DNA damage. OsUbc13 catalyzes K63 polyubiquitination in vitro,suggesting that it is a housekeeping gene.

Annotated Information

Function

Figure 1. Physical interaction of OsUbc13 with Mms2/Uev1A from other species.

Figure 4. In vitro ubiquitin conjugation assays using purified proteins.

OsUBC13 Functionally complements yeast ubc13 null mutant.OsUBC13 rescues the yeast ubc13 mutant from killing by MMS (alkylation damage), 4-nitroquinoline 1-oxide (4NQO, bulky lesions) and UV irradiation to a level comparable to that in wild-type cells.

OsUbc13 Physically interacts with yeast and human E2 variants((hUev1A and hMms2, yMms2 ).in pull-down assay (Figure 1). 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.all three Uevs from human and yeast can form stable heterodimers with OsUbc13 in vitro and in vivo.

OsUbc13, hUev1A and hMms2 were purified for an in vitro ubiquitination assay. As shown in Figure4, OsUbc13 (Lane 1), hUev1A (Lane 3) and hMms2 (Lane 7) alone did not promote free poly-ubiquitin chain formation. When both OsUbc13 and a Uev (hUev1A or hMms2) were present in the same reaction, di-Ub, tri-Ub and even longer Ub chains were readily formed (Lanes 4 and 8). Moreover, the assembly of free poly-Ub chains was not affected by the Ub-K48R mutation (Lanes 5 and 9); however, the Ub-K63R mutation completely abolished the Ub chain formation (Lanes 6 and 10). The above results indicate that with the assistance of hUev1A or hMms2, OsUbc13, can catalyze free poly-ubiquitin chain formation through K63 linkage.

Expression

Figure 2. Quantitative analyses of OsUBC13 expression.

the expression of OsUBC13 (LOC_Os01g48280) remains high and remarkably stable during development (Figure 2) and in different tissues.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 2b and quantitated by densitometry (data not shown), no obvious alteration was observed by any given treatments under our experimental conditions.

Evolution

Figure 3. Sequence analysis of OsUbc13 and its gene structure.

OsUbc13 also shows a high degree of conservation with Ubc13s from other eukaryotic organisms. Moreover, critical amino acid residues defined in hUbc13, including Cys87 in the active site for Ub thioester formation , Met64 for the physical interaction with RING finger E3 ligases ,Glu55, Phe57 and Arg70, which form a “pocket” that determine the specific interaction with Mms2 , as well as a Q100 residue as the target of bacterial OspI deamidation, are all conserved in OsUbc13 (Figure 3a).

Labs working on this gene

Wei Xiao. Department of Microbiology and Immunology, University of Saskatchewan, Saskatoon, SK, S7N 5E5, Canada.Email: wei.xiao@usask.ca

References

Zang et al.;Rice UBC13, a candidate housekeeping gene, is required for K63-linked polyubiquitination and tolerance to DNA damage
 Rice, 2012, 5: 24