A SINGLE HMG DOMAIN IN HIGH-MOBILITY GROUP-1 PROTEIN BINDS TO DNAS ASSMALL AS 20 BASE-PAIRS CONTAINING THE MAJOR CISPLATIN ADDUCT

Proteins containing a relatively new DNA-binding motif known as the hi gh-mobility group (HMG) domain bind specifically to DNA modified by th e anticancer drug cisplatin, but not to unmodified DNA (McA'Nulty & Li ppard, 1995). Southwestern-blot analyses of the binding of proteolytic fragments of HMG1 to a 123-bp globally platinated DNA demonstrate tha t the HMG domains A and B of HMG1 are responsible for its specific int eractions with cisplatin-modified DNA. An 81 amino acid recombinant pr otein representing a single HMG motif, HMG1 domain B, binds with an af finity (Kd = 10(-7) M) equal to that of HMG1 itself to 92- and 100-bp DNAs containing the major adduct of cisplatin, a cis-[Pt(NH3)(2)-{d(Gp G)-N7(1), -N7(2)}] intrastrand cross-link, at a specific site. The iso lated HMG domain B binds with comparable affinity to cisplatin-modifie d DNAs having as few as 20 bp. The related human mitochondrial HMG dom ain protein mtTFA also recognizes the 123-bp globally platinated DNA, providing further evidence that HMG domains are responsible for modula ting binding of this class of proteins to cisplatin-modified DNA. This work provides direct biochemical evidence in support of conclusions d rawn previously from analyses of sequence conservation (Bruhn et al., 1992) that HMG domains are the key elements in protein binding to cisp latin-modified DNA.