Ubiquitin-associated (UBA) domains in Rad23 bind ubiquitin and promote inhibition of multi-ubiquitin chain assembly.

Rad23 is a DNA repair protein that promotes the assembly of the nucleotide excision repair complex. Rad23 can interact with the 26S proteasome through an N-terminal ubiquitin-like domain, and inhibits the assembly of substrate-linked multi-ubiquitin (multi-Ub) chains in vitro and in vivo. Significantly, Rad23 can bind a proteolytic substrate that is conjugated to a few ubiquitin (Ub) moieties. We report here that two ubiquitin-associated (UBA) domains in Rad23 form non-covalent interactions with Ub. A mutant that lacked either UBA sequence was capable of blocking the assembly of substrate-linked multi-Ub chains, although a mutant that lacked both UBA domains was significantly impaired. These studies suggest that the interaction with Ub is required for Rad23 activity, and that other UBA-containing proteins may have a similar function.

The DNA repair protein rad23 is a negative regulator of multi-ubiquitin chain assembly.

Rad23 is a nucleotide-excision repair protein with a previously unknown biochemical function. We determined that yeast and human Rad23 inhibited multi-ubiquitin (Ub) chain formation and the degradation of proteolytic substrates. Significantly, Rad23 could be co-precipitated with a substrate that contained a short multi-Ub chain. The UV sensitivity of rad23Delta was reduced in mutants lacking the E2 enzyme Ubc4, or the multi-Ub chain-promoting factor Ufd2. These studies suggest that the stability of proteolytic substrates is governed by the competing action of multi-Ub chain-promoting and chain-inhibiting factors. The stabilization of DNA repair and stress factors could represent an important biological function of Rad23.