We have determined the effect of HMG-1 bound to cisplatin-damaged DNA on the activities of calf helicase E. DNase I protection analysis demonstrated HMG-1 bound a cisplatin-damaged 24 base oligonucleotide annealed to M13mp18. Exonuclease digestion experiments revealed that greater than 90% of the DNA substrates contained a single site specific cisplatin adduct and, maximally, 65% of the substrates were bound by HMG-1. Helicase E catalyzed displacement of the cisplatin-damaged DNA oligonucleotide was inhibited by HMG-1 in a concentration-dependent manner. Time course experiments revealed a decreased rate of displacement in reactions containing HMG-1. The maximum inhibition observed was 55% and taking into account that only 65% of the substrates had HMG-1 bound, approximately 85% inhibition was observed on platinated DNA substrates containing HMG-1. Inhibition of helicase activity was proportional to the amount of substrate bound by HMG-1 based on the displacement and exonuclease assays at varying HMG-1 concentrations. The ability of helicase E to displace an undamaged DNA oligonucleotide from a cisplatin-damaged DNA template was also inhibited by HMG-1. Interestingly, HMG-1 had no effect on the rate of DNA-dependent ATP hydrolysis catalyzed by helicase E on the same DNA substrate. The inhibition of helicase activity by HMG-1 binding cisplatin-damaged DNA further supports a role for HMG-1 inhibiting DNA repair which may contribute to cellular sensitivity to cisplatin.