Photo-Affinity Isolation and Identification of Proteins in Cancer Cell Extracts that Bind to Platinum-Modified DNA

摘要

The activity of the anticancer drug cisplatin is a consequence of its ability to bind DNA. Platinum adducts bend and unwind DNA duplex, creating recognition sites for nuclear proteins. Following DNA damage recognition, the lesions will either be repaired, facilitating cell viability, or if repair is unsuccessful and the Pt adduct interrupts vital cellular functions, apoptosis will follow. With the use of the benzophenone-modified cisplatin analogue Pt-BP6, 25-bp DNA duplexes containing either a 1,2-d(G*pG*) intrastrand or a 1,3-d(G*pTpG*) intrastrand cross-link were synthesized, where the asterisks designate platinated nucleobases. Proteins having affinity for these platinated DNAs were photo-cross-linked and identified in cervical, testicular, pancreatic and bone cancer cell nuclear extracts. Proteins identified in this manner include the DNA repair factors RPA1, Ku70, Ku80, Msh2, DNA ligase III, PARP-1, and DNA-PKcs, as well as HMG-domain proteins HMGB1, HMGB2, HMGB3, and UBF1. The latter strongly associate with the 1,2-d(G*pG*) adduct and weakly or not at all with the 1,3-d(G*pTpG*) adduct. The nucleotide excision repair protein RPA1 was photo-cross-linked only by the probe containing a 1,3-d(G*pTpG*) intrastrand cross-link. The affinity of PARP-1 for platinum-modified DNA was established using this type of probe for the first time. To ensure that the proteins were not photo-cross-linked because of an affinity for DNA ends, a 90-base dumbbell probe modified with Pt-BP6 was investigated. Photo-cross-linking experiments using this longer probe revealed the same proteins, as well as some additional proteins involved in chromatin remodeling, transcription, or repair. These findings reveal a more complete list of proteins involved in the early step of the mechanism of action of the cisplatin and its close analogue carboplatin.