THE HMG-DOMAIN PROTEIN IXR1 BLOCKS EXCISION-REPAIR OF CISPLATIN-DNA ADDUCTS IN YEAST

Ixr1 is a yeast HMG-domain protein which binds the major DNA adducts o f the antitumor drug cisplatin. Previous work demonstrated that Saccha romyces cerevisiae cells lacking the IXR1 gene were two-fold less sens itive to cisplatin treatment than wild-type cells, and the present inv estigation reveals a six-fold difference in yeast having a different b ackground. The possibility that the lower cytotoxicity of cisplatin in the ixr1 strain is the result of enhanced repair was investigated in rad1, rad2, rad4, rad6 rad9, rad10, rad14 and rad52 backgrounds. In th ree of the excision repair mutants, rad2, rad4 and rad14, the differen tial sensitivity caused by removing the Ixr1 protein was nearly abolis hed. This result demonstrates that the greater cisplatin resistance in the ixr1 strain is most likely a consequence of excision repair, supp orting the theory that Ixr1 and other HMG-domain proteins can block re pair of the major cisplatin-DNA adducts in vivo. The differential sens itivity of wild-type cells and those lacking Ixr1 persisted in the mdl and rad10 strains, however, indicating that these two proteins act at a stage in the excision repair pathway where damage recognition is le ss critical. A model is proposed to account for these results, which i s strongly supported recently identified functional roles for the rad excision repair gene products. A rad52 mutant was more sensitive to ci splatin than the RAD52 parental strain, which reveals that Rad52, a do uble-strand break repair protein, repairs cisplatin-DNA adducts, proba bly interstrand cross-links. A rad52 ixr1 strain was less sensitive to cisplatin than the rad52 IXR1 strain, consistent with Ixr1 not blocki ng repair of cisplatin adducts removed by Rad52. rad6 strains behaved similarly, except they were both substantially more sensitive to cispl atin. Interruption of the RAD9 gene, which is involved in DNA-damage-i nduced cell cycle arrest, had no affect on cisplatin cytotoxicity.