title: Interaction of FACT, SSRP1, and the high mobility group (HMG) domain of SSRP1 with DNA damaged by the anticancer drug cisplatin.
abstract: The structure-specific recognition protein SSRP1, initially isolated from expression screening of a human B-cell cDNA library for proteins that bind to cisplatin (cis-diamminedichloroplatinum(II))-modified DNA, contains a single DNA-binding high mobility group (HMG) domain. Human SSRP1 purifies as a heterodimer of SSRP1 and Spt16 (FACT) that alleviates the nucleosomal block to transcription elongation by RNAPII in vitro. The affinity and specificity of FACT, SSRP1, and the isolated HMG domain of SSRP1 for cisplatin-damaged DNA were investigated by gel mobility shift assays. FACT exhibits both affinity and specificity for DNA damaged globally with cisplatin compared with unmodified DNA or DNA damaged globally with the clinically ineffective trans-DDP isomer. FACT binds the major 1,2-d(GpG) intrastrand cisplatin adduct, but its isolated SSRP1 subunit fails to form discrete, high affinity complexes with cisplatin-modified DNA under similar conditions. These results 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 major cisplatin 1,2-d(GpG) intrastrand cross-link. The affinity and specificity of FACT for cisplatin-modified DNA, as well as its importance for transcription of chromatin, suggests that the interaction of FACT and cisplatin-damaged DNA may be crucial to the anticancer mechanism of cisplatin.
authors.affiliation: Department of Chemistry, Massachusetts Institute of Technology, Cambridge, MA 02139, USA.
authors.forename: A T
authors.initials: AT
authors.lastname: Yarnell
authors.forename: S
authors.initials: S
authors.lastname: Oh
authors.forename: D
authors.initials: D
authors.lastname: Reinberg
authors.forename: S J
authors.initials: SJ
authors.lastname: Lippard
mesh_terms: D000595:Amino Acid Sequence; D000970:Antineoplastic Agents / Q000494:pharmacology*; D002945:Cisplatin / Q000494:pharmacology*; D004249:DNA Damage / Q000187:drug effects*; D004268:DNA-Binding Proteins / Q000235:genetics*; D015972:Gene Expression Regulation, Neoplastic / Q000187:drug effects; D006367:HeLa Cells; D006609:High Mobility Group Proteins / Q000235:genetics*; D006801:Humans; D008969:Molecular Sequence Data; D009369:Neoplasms / Q000188:drug therapy / Q000235:genetics; D014157:Transcription Factors / Q000235:genetics*; D035602:Transcriptional Elongation Factors
chemical_list: D000970:Antineoplastic Agents; D004268:DNA-Binding Proteins; D006609:High Mobility Group Proteins; C073537:SSRP1 protein, human; D014157:Transcription Factors; D035602:Transcriptional Elongation Factors; D002945:Cisplatin