Nucleotide Excision Repair from Site-Specifically Platinum-Modified Nucleosomes ² Dong Wang, ‡ Ryujiro Hara, § Gitanjali Singh, ‡ Aziz Sancar, § and Stephen J. Lippard* ,‡ Department of Chemistry, Massachusetts Institute of Technology, Cambridge, Massachusetts 02139, and Department of Biochemistry and Biophysics, UniVersity of North Carolina, School of Medicine, Chapel Hill, North Carolina 27599 ReceiVed February 17, 2003 ABSTRACT: Nucleotide excision repair is a major cellular defense mechanism against the toxic effects of the anticancer drug cisplatin and other platinum-based chemotherapeutic agents. In this study, mononu- cleosomes were prepared containing either a site-specific cis-diammineplatinum(II)-DNA intrastrand d(GpG) or a d(GpTpG) cross-link. The ability of the histone core to modulate the excision of these defined platinum adducts was investigated as a model for exploring the cellular response to platinum-DNA adducts in chromatin. Comparison of the extent of repair by mammalian cell extracts of free and nucleosomal DNA containing the same platinum-DNA adduct reveals that the nucleosome significantly inhibits nucleotide excision repair. With the GTG-Pt DNA substrate, the nucleosome inhibits excision to about 10% of the level observed with free DNA, whereas with the less efficient GG-Pt DNA substrate the nucleosome inhibited excision to about 30% of the level observed with free DNA. The effects of post- translational modification of histones on excision of platinum damage from nucleosomes were investigated by comparing native and recombinant nucleosomes containing the same intrastrand d(GpTpG) cross-link. Excision from native nucleosomal DNA is ∼2-fold higher than the level observed with recombinant material. This result reveals that post-translational modification of histones can modulate nucleotide excision repair from damaged chromatin. The in vitro system established in this study will facilitate the investigation of platinum-DNA damage by DNA repair processes and help elucidate the role of specific post-translational modification in NER of platinum-DNA adducts at the physiologically relevant nucleosome level. Cisplatin, cis-diamminedichloroplatinum(II), and other platinum-based drugs such as carboplatin, cis-diammine(1,1- cyclobutanedicarboxylato)platinum(II) (Figure 1), are used to treat testicular tumors as well as a variety of other cancers (1, 2). DNA is the principal cellular target of these compounds (3), the platinum atom forming covalent bonds to the N7 positions of the purine bases. The major adducts are 1,2-intrastrand and 1,3-intrastrand cross-links (4-6). Although cisplatin and carboplatin both form these identical bifunctional adducts, the relative frequency of individual platinum-DNA cross-links is quite different. For cisplatin, 1,2-intrastrand adducts comprise 50-90%, and 1,3-intra- strand 10-25%, of all DNA lesions in cultured Chinese hamster ovary (CHO) 1 cells treated with the drug. However, the percentage of 1,2- and 1,3-intranstrand cross-links for carboplatin treatment of the same CHO cells is 35-50% and 30-40%, respectively (6). Since carboplatin is widely used in cancer chemotherapy, it is important to study both the 1,2- and 1,3-intrastrand cis-diammineplatinum(II) cross-links formed by the two compounds. The cellular events that are triggered by platinum-DNA damage are the subject of considerable interest, and detailed knowledge of these processes could facilitate the rational design of better platinum-based drugs. For example, under- standing how platinum compounds with different ligands block DNA repair and transcription more efficiently may suggest a strategy for designing new drugs. Cells deficient ² This work was supported by National Cancer Institute Grant CA34992 (S.J.L) and the National Institute of General Medical Sciences GM32833 (A.S.). D.W. is a predoctoral fellow of the Anna Fuller Foundation, and G.S. was supported under the MIT Undergraduate Research Opportunity Program. * To whom correspondence should be addressed. E-mail: lippard@ lippard.mit.edu. Telephone: (617) 253-1892. Fax: (617) 258-8150. ‡ Massachusetts Institute of Technology. § University of North Carolina. 1 Abbreviations: NER, nucleotide excision repair; CHO, Chinese hamster ovary; XPA, xeroderma pigmentosum complementation group A; XPF, xeroderma pigmentosum complementation group F; ERCC, excision repair cross complementing; GTG-Pt, DNA probe containing a site-specific intrastrand cisplatin cross-link at a single d(GpTpG) site; GG-Pt, DNA probe containing a site-specific intrastrand cisplatin cross- link at a single d(GpG) site; PMSF, phenylmethylsulfonyl fluoride; DTT, dithiothreitol; bp, base pair(s); IE HPLC, ion exchange high- performance liquid chromatography; AAS, atomic absorption spec- troscopy; PAGE, polyacrylamide gel electrophoresis; TBE, Tris-borate- EDTA; BSA, bovine serum albumin. FIGURE 1: Structures of cisplatin and carboplatin. (A) Cisplatin, cis-diamminedichloroplatinum(II). (B) Carboplatin, cis-diammine- (1,1-cyclobutanedicarboxylato)platinum(II). 6747 Biochemistry 2003, 42, 6747-6753 10.1021/bi034264k CCC: $25.00 © 2003 American Chemical Society Published on Web 05/14/2003