UBE2B (HR6B) [6His-tagged]

Ubiquigent

Catalog No: UBI-62-0072-020

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Species Reactivity: Human  
Source: E. coli expression  
Formula: 50 mM HEPES pH 7.5, 150 mM sodium chloride, 2 mM dithiothreitol, 10% glycerol  
Concentration: 1 mg/ml  
Molecular Weight: ~20 kDa  
Protein Sequence: Accession number: NP_003328. For full protein sequence information download the Certificate of Analysis pdf.  
Quality Control Protein Identification: Confirmed by mass spectrometry.  
Quality Control Activity: E2-Ubiquitin Thioester Loading Assay:  The activity of His-UBE2B was validated by loading E1 UBE1 activated ubiquitin onto the active cysteine of the His-UBE2B E2 enzyme via a transthiolation reaction. Incubation of the UBE1 and His-UBE2B enzymes in the presence of ubiquitin and ATP at 30 °C was compared at two time points, T0  and T10  minutes. Sensitivity of the ubiquitin/His-UBE2B thioester bond to the reducing agent DTT was confirmed.  

Background  

The enzymes of the ubiquitylation pathway play a pivotal role in a number of cellular processes including regulated and targeted proteasomal degradation of substrate proteins. Three classes of enzymes are involved in the process of ubiquitylation; activating enzymes (E1s), conjugating enzymes (E2s) and protein ligases (E3s). UBE2B is a member of the E2 ubiquitin-conjugating enzyme family and cloning of the human gene was first described by Koken et al. (1991). UBE2B shares 70% identity with its yeast homologue but lacks the acidic C-terminal domain. The ring finger proteins RAD5 and RAD18 interact with UBE2B and other members of the RAD6 pathway (Notenboom et al., 2007; Ulrich and Jentsch, 2000). In complex UBE2B and RAD18 trigger replication fork stalling at DNA damage sites during the post replicative repair process (Tsuji et al., 2008). Null mutations of the UBE2B gene in mice are associated with structural abnormalities in sperm and SNP analysis of human UBE2B variants has provided evidence for association of this gene with male infertility (Escalier et al., 2003; Suryavathi et al., 2008).

References:

Escalier D, Bai XY, Silvius D, Xu PX, Xu X (2003) Spermatid nuclear and sperm periaxonemal anomalies in the mouse Ube2b null mutant. Mol Reprod Dev 65, 298-308.

Koken MH, Reynolds P, Jaspers-Dekker I, Prakash L, Prakash S, Bootsma D, Hoeijmakers JH (1991) Structural and functional conservation of two human homologs of the yeast DNA repair gene RAD6. Proc Natl Acad Sci U S A 88, 8865-9.

Notenboom V, Hibbert RG, van Rossum-Fikkert SE, Olsen JV, Mann M, Sixma TK (2007) Functional characterization of Rad18 domains for Rad6, ubiquitin, DNA binding and PCNA modification. Nucleic Acids Res 35, 5819-30.

Suryavathi V, Khattri A, et al. (2008) Novel variants in UBE2B gene and idiopathic male infertility. J Androl 29, 564-71.

Tsuji Y, Watanabe K, et al. (2008) Recognition of forked and single-stranded DNA structures by human RAD18 complexed with RAD6B protein triggers its recruitment to stalled replication forks. Genes Cells 13, 343-54.

Ulrich HD, Jentsch S (2000) Two RING finger proteins mediate cooperation between ubiquitin-conjugating enzymes in DNA repair. EMBO J 19, 3388-97.