RING (really interesting new gene) and U-box E3 ligases bridge E2 
ubiquitin-conjugating enzymes and substrates to enable the transfer of ubiquitin 
to a lysine residue on the substrate or to one of the seven lysine residues of 
ubiquitin for polyubiquitin chain elongation. Different polyubiquitin chains 
have different functions. Lys(48)-linked chains target proteins for proteasomal 
degradation, and Lys(63)-linked chains function in signal transduction, 
endocytosis and DNA repair. For this reason, chain topology must be tightly 
controlled. Using the U-box E3 ligase CHIP [C-terminus of the Hsc (heat-shock 
cognate) 70-interacting protein] and the RING E3 ligase TRAF6 
(tumour-necrosis-factor-receptor-associated factor 6) with the E2s Ubc13 
(ubiquitin-conjugating enzyme 13)-Uev1a (ubiquitin E2 variant 1a) and UbcH5a, in 
the present study we demonstrate that Ubc13-Uev1a supports the formation of free 
Lys(63)-linked polyubiquitin chains not attached to CHIP or TRAF6, whereas 
UbcH5a catalyses the formation of polyubiquitin chains linked to CHIP and TRAF6 
that lack specificity for any lysine residue of ubiquitin. Therefore the 
abilities of these E2s to ubiquitinate a substrate and to elongate polyubiquitin 
chains of a specific topology appear to be mutually exclusive. Thus two 
different classes of E2 may be required to attach a polyubiquitin chain of a 
particular topology to a substrate: the properties of one E2 are designed to 
mono-ubiquitinate a substrate with no or little inherent specificity for an 
acceptor lysine residue, whereas the properties of the second E2 are tailored to 
the elongation of a polyubiquitin chain using a defined lysine residue of 
ubiquitin.