Interaction of FACT, SSRP1, and the high mobility group (HMG) domain of SSRP1 with DNA damaged by the anticancer drug cisplatin

The structure-specific recognition protein SSRP1, initially isolated from e xpression screening of a human B-cell cDNA library for proteins that bind t o cisplatin (cis-diamminedichloroplatinum(II))-modified DNA, contains a sin gle DNA-binding high mobility group (HMG) domain. Human SSRP1 purifies as a heterodimer of SSRP1 and Spt16 (FACT) that alleviates the nucleosomal bloc k to transcription elongation by RNAPII in vitro. The affinity and specific ity of FACT, SSRP1, and the isolated HMG domain of SSRP1 for cisplatin-dama ged DNA were investigated by gel mobility shift assays, FACT exhibits both affinity and specificity for DNA damaged globally with cisplatin compared w ith unmodified DNA or DNA damaged globally with the clinically ineffective trans-DDP isomer, FACT binds the major 1,2-d(GpG) intrastrand cisplatin add uct, but its isolated SSRP1 subunit fails to form discrete, high affinity c omplexes with cisplatin-modified DNA under similar conditions. These result s suggest that Spt16 primes SSRP1 for cisplatin-damaged DNA recognition by unveiling its HMG domain, As expected, the isolated HMG domain of SSRP1 is sufficient for specific binding to cisplatin-damaged DNA and binds the majo r cisplatin 1,1-d(GpG) intrastrand cross-link, The affinity and specificity of FACT for cisplatin-modified DNA as well as its importance for transcrip tion of chromatin, suggests that the interaction of FACT and cisplatin-dama ged DNA may be crucial to the anticancer mechanism of cisplatin.