Photoaffinity labeling reveals nuclear proteins that uniquely recognize cisplatin-DNA interstrand cross-links.
The DNA-binding inorganic compound cisplatin is one of the most successful anticancer drugs. The detailed mechanism by which cells recognize and process cisplatin-DNA damage is of great interest. Although the family of proteins that bind cisplatin 1,2- and 1,3-intrastrand cross-links has been identified, much less is known about cellular protein interactions with cisplatin interstrand cross-links (ICLs). In order to address this question, a photoreactive analogue of cisplatin, PtBP(6), was used to construct a DNA duplex containing a site-specific platinum ICL. This DNA probe was characterized and used in photo-cross-linking experiments to separate and identify nuclear proteins that bind to the ICL by peptide mass fingerprint analysis. Several such proteins were discovered, including PARP-1, hMutSbeta, DNA ligase III, XRCC1, and PNK. The photo-cross-linking approach was independently validated by an electrophoretic mobility shift assay demonstrating hMutSbeta binding to a cisplatin ICL. Proteins that recognize the platinum ICL were also identified in cisplatin-resistant cells, cells halted at various phases of the cell cycle, and in different carcinoma cells. Nuclear proteins that bind to the platinum ICL differ from those binding to intrastrand cross-links, indicating different mechanisms for disruption of cellular functions.
Department of Chemistry, Massachusetts Institute of Technology, Cambridge, Massachusetts 02139, USA.
Guangyu
G
Zhu
Stephen J
SJ
Lippard
D002945:Cisplatin / Q000031:analogs & derivatives / Q000378:metabolism*; D003432:Cross-Linking Reagents / Q000138:chemical synthesis / Q000378:metabolism*; D018736:DNA Adducts / Q000737:chemistry / Q000378:metabolism*; D015342:DNA Probes / Q000138:chemical synthesis / Q000378:metabolism*; D004260:DNA Repair / Q000235:genetics; D006367:HeLa Cells; D006801:Humans; D009687:Nuclear Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D009692:Nucleic Acid Heteroduplexes / Q000138:chemical synthesis / Q000378:metabolism; D019920:Photoaffinity Labels / Q000378:metabolism*; D040081:Protein Array Analysis / Q000379:methods; D011485:Protein Binding / Q000235:genetics; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; D053719:Tandem Mass Spectrometry
D003432:Cross-Linking Reagents; D018736:DNA Adducts; D015342:DNA Probes; D009687:Nuclear Proteins; D009692:Nucleic Acid Heteroduplexes; D019920:Photoaffinity Labels; D002945:Cisplatin