Electrospray ionization-mass spectrometry study of the interaction of cisplatin-adducted oligonucleotides with human XPA minimal binding domain protein

Nucleotide excision repair (NER) is the process responsible for eliminating most ultraviolet (UV) radiation damage from DNA, as well as base alteratio ns caused by a variety of mutagens. The xeroderma pigmentosum group A compl ementing protein (XPA) is believed to be involved in the early step of NER by recognizing and binding damaged DNA. Recent work has suggested that elec trospray ionization-mass spectrometry (ESI-MS) can be an effective tool for the study of protein-DNA complexes. We have used ESI-Fourier transform ion cyclotron resonance (FTICR) mass spectrometry to examine the cisplatin-add ucted oligonucleotide and its interaction with the human XPA minimal bindin g domain (XPA-MBD). High-resolution FTICR experiments of the binding produc ts showed that both double-stranded damaged 20-mer and double-stranded unda maged 20-mer formed 1:1 noncovalent complexes with XPA-MBD. A 2:1 binding s toichiometry complex was also observed between XPA-MBD and double-stranded damaged 20-mer. Competitive binding experiments indicated only slightly pre ferential binding of XPA-MBD with the double-stranded damaged 20-mer compar ed to the undamaged 20-mer. The results demonstrate that ESI-FTICR mass spe ctrometry provides a fast and efficient approach for characterizing weak pr otein-DNA interactions such as the binding between XPA-MBD and a 20-mer oli gonucleotide system. (C) 1999 Academic Press.