title: Nucleotide excision repair from site-specifically platinum-modified nucleosomes.
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, mononucleosomes 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 approximately 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.
authors.affiliation: Department of Chemistry, Massachusetts Institute of Technology, Cambridge, Massachusetts 02139, USA.
authors.forename: Dong
authors.initials: D
authors.lastname: Wang
authors.forename: Ryujiro
authors.initials: R
authors.lastname: Hara
authors.forename: Gitanjali
authors.initials: G
authors.lastname: Singh
authors.forename: Aziz
authors.initials: A
authors.lastname: Sancar
authors.forename: Stephen J
authors.initials: SJ
authors.lastname: Lippard
mesh_terms: D000818:Animals; D001483:Base Sequence; D016466:CHO Cells; D002945:Cisplatin / Q000737:chemistry*; D006224:Cricetinae; D018736:DNA Adducts / Q000737:chemistry; D015342:DNA Probes / Q000235:genetics; D004260:DNA Repair; D004591:Electrophoresis, Polyacrylamide Gel / Q000379:methods; D006657:Histones / Q000235:genetics / Q000378:metabolism; D008969:Molecular Sequence Data; D009707:Nucleosomes / Q000737:chemistry* / Q000235:genetics; D009838:Oligodeoxyribonucleotides / Q000737:chemistry / Q000235:genetics; D010759:Phosphorus Isotopes; D011499:Protein Processing, Post-Translational; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism
chemical_list: D018736:DNA Adducts; D015342:DNA Probes; D006657:Histones; D009707:Nucleosomes; D009838:Oligodeoxyribonucleotides; D010759:Phosphorus Isotopes; D011994:Recombinant Proteins; D002945:Cisplatin