YEAST DNA-REPAIR PROTEINS RAD6 AND RAD18 FORM A HETERODIMER THAT HAS UBIQUITIN-CONJUGATING, DNA-BINDING, AND ATP HYDROLYTIC ACTIVITIES

The RAD6 and RAD18 genes of Saccharomyces cerevisiae are required far postreplicative bypass of ultraviolet (UV)-damaged DNA and for UV muta genesis. The RAD6 encoded protein is a ubiquitin conjugating enzyme, a nd RAD18 encodes a protein containing a RING finger motif and a nucleo tide binding motif, Rad18 can be co-immunoprecipitated with Rad6, indi cating that the two proteins exist in a complex in vivo. Here, we co-o verproduce the two proteins using a yeast multicopy plasmid, purify th e Rad6-Rad18 complex to near homogeneity, and show that the complex is heterodimeric. The Rad6-Rad18 heterodimer has ubiquitin conjugating a ctivity, binds single-stranded DNA, and possesses single-stranded DNA- dependent ATPase activity. The Rad6-Rad18 complex provides the first e xample wherein a ubiquitin conjugating activity is physically associat ed with DNA binding and ATPase activities provided by an associated pr otein factor, The co-existence of these activities should provide the complex with the ability to recognize single-stranded DNA resulting fr om stalling of the replication machinery at DNA damage sites and to re cognize the components of the DNA replication machinery for ubiquitina tion by Rad6.