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 substrat e-linked multiubiquitin (multi-Ub) chains in vitro and in vivo. Significant ly, Rad23 can bind a proteolytic substrate that is conjugated to a few ubiq uitin (Ub) moieties. We report here that two ubiquitin-associated (UBA) dom ains in Rad23 form non-covalent interactions with Ub. A mutant that lacked either UBA sequence was capable of blocking the assembly of substrate-linke d multi-Ub chains, although a mutant that lacked both UBA domains was signi ficantly impaired. These studies suggest that the interaction with Ub is re quired for Rad23 activity and that other UBA-containing proteins may have a similar function.