BINDING OF IXR1, A YEAST HMG-DOMAIN PROTEIN, TO CISPLATIN-DNA ADDUCTSIN-VITRO AND IN-VIVO

Ixr1 is a yeast HMG-domain protein that binds specifically to DNA addu cts formed by the antitumor drug cisplatin. interruption of the IXR1 g ene in yeast desensitizes cells to cisplatin. This effect is unrelated to a natural function of Ixr1, which is to repress the transcription of COX5b. Ixr1 interacts specifically and preferentially with DNA modi fied by cisplatin. In the present work, Ixr1 was purified from a clone expressed in Escherichia coli. The dissociation constant for Ixr1 bin ding site-specifically to a 92-bp probe containing a single cis-[Pt(NH 3)(2){d(GpG)-N7(1) -N7(2)}] intrastrand cross-link was measured to be 2.5 (+/-10.1) x 10(-7) M, similar to that found for HMG1. Ixr1 binds a t least an order of magnitude more tightly to cisplatin-DNA adducts th an to unmodified DNA. Hydroxyl radical footprinting revealed that Ixr1 protects an area of platinated DNA that is approximately 15 bp in siz e and centered at the platinum adduct. The binding of HMG-domain prote ins to cisplatin-DNA adducts has been proposed to divert these protein s from their natural DNA-binding sites, disrupting transcription. This hypothesis was tested for Ixr1 in yeast. The protein was not titrated away from the Cox5b promoter sufficiently well to disrupt transcripti on either of Cox5b mRNA from genomic DNA or of the beta-galactosidase gene under control of the promoter in a plasmid DNA transformed into y east.