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 alterations caused by a variety of mutagens. The xeroderma pigmentosum group A complementing protein (XPA) is believed to be involved in the early step of NER by recognizing and binding damaged DNA. Recent work has suggested that electrospray 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-adducted oligonucleotide and its interaction with the human XPA minimal binding domain (XPA-MBD). High-resolution FTICR experiments of the binding products showed that both double-stranded damaged 20-mer and double-stranded undamaged 20-mer formed 1:1 noncovalent complexes with XPA-MBD. A 2:1 binding stoichiometry complex was also observed between XPA-MBD and double-stranded damaged 20-mer. Competitive binding experiments indicated only slightly preferential binding of XPA-MBD with the double-stranded damaged 20-mer compared to the undamaged 20-mer. The results demonstrate that ESI-FTICR mass spectrometry provides a fast and efficient approach for characterizing weak protein-DNA interactions such as the binding between XPA-MBD and a 20-mer oligonucleotide system.
Xu
N
N
Environmental Molecular Sciences Laboratory, Pacific Northwest National Laboratory, Richland, Washington 99352, USA.
Pasa-Tolić
L
L
Smith
R D
RD
Ni
S
S
Thrall
B D
BD
D001483:Base Sequence; D001667:Binding, Competitive; D002945:Cisplatin / Q000737:chemistry* / Q000378:metabolism; D003432:Cross-Linking Reagents / Q000737:chemistry / Q000378:metabolism; D018736:DNA Adducts / Q000737:chemistry* / Q000378:metabolism; D004249:DNA Damage; D004260:DNA Repair; D004268:DNA-Binding Proteins / Q000737:chemistry* / Q000378:metabolism; D006801:Humans; D066298:In Vitro Techniques; D013058:Mass Spectrometry / Q000379:methods*; D009838:Oligodeoxyribonucleotides / Q000737:chemistry / Q000378:metabolism; D010446:Peptide Fragments / Q000737:chemistry / Q000378:metabolism; D011485:Protein Binding; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D051760:Xeroderma Pigmentosum Group A Protein
D003432:Cross-Linking Reagents; D018736:DNA Adducts; D004268:DNA-Binding Proteins; D009838:Oligodeoxyribonucleotides; D010446:Peptide Fragments; D011994:Recombinant Proteins; C497314:XPA protein, human; D051760:Xeroderma Pigmentosum Group A Protein; D002945:Cisplatin