SPECIFIC COMPLEX-FORMATION BETWEEN YEAST RAD6 AND RAD18 PROTEINS - A POTENTIAL MECHANISM FOR TARGETING RAD6 UBIQUITIN-CONJUGATING ACTIVITY TO DNA-DAMAGE SITES

The RAD6 gene of Saccharomyces cerevisiae encodes a ubiquitin-conjugat ing enzyme that is required for postreplication repair of UV damaged D NA, DNA damage induced mutagenesis, sporulation, and amino-end rule pr otein degradation. RAD6 interacts physically with the UBR1 gene produc t in carrying out the multiubiquitination of amino-end rule proteolyti c substrates. In mediating postreplication repair, it has remained unc lear whether RAD6 acts in a pleiotropic manner distal from the site of DNA damage or is targeted to the damage site via interaction with ano ther repair component. Here, we show that RAD6 forms a specific comple x with the product of the DNA repair gene RAD18. The biological signif icance of this interaction is attested by the observation that overpro duction of the rad6 Ala-88 mutant protein, which lacks ubiquitin-conju gating activity but retains the ability to interact with RAD18 protein , confers a high level of UV sensitivity on wild-type RAD(+) cells tha t can be corrected by the concomitant overexpression of RAD18. We demo nstrate that whereas RAD6 has no affinity for DNA, RAD18 binds single- stranded DNA. Thus, association of RAD6 with RAD18 could provide a mea ns for targeting RAD6 to damage-containing DNA regions, where the RAD6 ubiquitin-conjugating function could modulate the activity of a stall ed DNA replication machinery. We also show that RAD6 forms separate co mplexes with RAD18 and with UBR1, and the extremely conserved amino te rminus of RAD6 that is required for complex formation with UBR1 is dis pensable for complex formation with RAD18.