title: Crosslinking of DNA repair and replication proteins to DNA in cells treated with 6-thioguanine and UVA.
abstract: The DNA of patients taking immunosuppressive and anti-inflammatory thiopurines contains 6-thioguanine (6-TG) and their skin is hypersensitive to ultraviolet A (UVA) radiation. DNA 6-TG absorbs UVA and generates reactive oxygen species that damage DNA and proteins. Here, we show that the DNA damage includes covalent DNA-protein crosslinks. An oligonucleotide containing a single 6-TG is photochemically crosslinked to cysteine-containing oligopeptides by low doses of UVA. Crosslinking is significantly more efficient if guanine sulphonate (G(SO3))--an oxidized 6-TG and a previously identified UVA photoproduct--replaces 6-TG, suggesting that G(SO3) is an important reaction intermediate. Crosslinking occurs via oligopeptide sulphydryl and free amino groups. The oligonucleotide-oligopeptide adducts are heat stable but are partially reversed by reducing treatments. UVA irradiation of human cells containing DNA 6-TG induces extensive heat- and reducing agent-resistant covalent DNA-protein crosslinks and diminishes the recovery of some DNA repair and replication proteins from nuclear extracts. DNA-protein crosslinked material has an altered buoyant density and can be purified by banding in cesium chloride (CsCl) gradients. PCNA, the MSH2 mismatch repair protein and the XPA nucleotide excision repair (NER) factor are among the proteins detectable in the DNA-crosslinked material. These findings suggest that the 6-TG/UVA combination might compromise DNA repair by sequestering essential proteins.
authors.affiliation: Cancer Research UK London Research Institute, Clare Hall Laboratories, South Mimms, EN6 3LD, UK.
authors.forename: Quentin
authors.initials: Q
authors.lastname: Gueranger
authors.forename: Azadeh
authors.initials: A
authors.lastname: Kia
authors.forename: David
authors.initials: D
authors.lastname: Frith
authors.forename: Peter
authors.initials: P
authors.lastname: Karran
mesh_terms: D004247:DNA / Q000737:chemistry* / Q000528:radiation effects; D004249:DNA Damage; D004260:DNA Repair; D004261:DNA Replication; D004268:DNA-Binding Proteins / Q000032:analysis / Q000737:chemistry*; D015180:Electrophoresis, Gel, Two-Dimensional; D006801:Humans; D051718:MutS Homolog 2 Protein / Q000032:analysis; D009687:Nuclear Proteins / Q000032:analysis / Q000737:chemistry*; D009842:Oligopeptides / Q000737:chemistry; D055668:Photochemical Processes; D018809:Proliferating Cell Nuclear Antigen / Q000032:analysis; D013866:Thioguanine / Q000737:chemistry*; D014407:Tumor Cells, Cultured; D014466:Ultraviolet Rays; D051760:Xeroderma Pigmentosum Group A Protein / Q000032:analysis
chemical_list: D004268:DNA-Binding Proteins; D009687:Nuclear Proteins; D009842:Oligopeptides; D018809:Proliferating Cell Nuclear Antigen; D051760:Xeroderma Pigmentosum Group A Protein; D004247:DNA; D051718:MutS Homolog 2 Protein; D013866:Thioguanine