Mass Spectrometric Analysis
of
UV-Cross-linked Protein-Nucleic Acid Complexes
Catalin E. Doneanu1, Philip R. Gafken2, Douglas F. Barofsky3,4
1Deparment of Medicinal Chemistry, University of Washington, Seattle, WA 98195
2Proteomics Facility, Fred Hutchinson Cancer Research Center, Seattle, WA 98109
3Department of Chemistry and the 4Environmental Health Sciences Center
Oregon State University, Corvallis, OR 97331
The study of the interaction of proteins, whether they are enzymes, regulatory proteins or structural proteins, with nucleic acids is one of the most exciting areas of modern molecular biology. Structural studies of DNA-binding proteins and their complexes with DNA have proceeded at an accelerated pace over the last two decades due to important technical advances in molecular genetics, DNA synthesis, UV-catalyzed cross-linking, protein crystallography, and nuclear magnetic resonance spectroscopy. The classical direct methods used to investigate protein-DNA interactions are X-ray diffraction and NMR spectroscopy, but both these methods have certain limitations.
The emergence of mass spectrometry as an analytical tool in biochemical research has opened up new possibilities for investigating protein-DNA interactions. It has been shown that intact nucleoprotein complexes can be analyzed by MALDI [1] and electrospray mass spectrometry [2].
A new direct method for studying protein-DNA complexes combining photochemical cross-linking with mass spectrometry was introduced by Barofsky et al in 1994 [3]. This method involves the purification and mass spectrometric analysis of nucleopeptide-products isolated from a tryptically digested UV-cross-linked protein-nucleic acid complex. Nucleopeptides were purified according to a general protocol and fragmented by tandem mass spectrometry in order to identify specific aminoacids involved in binding the DNA substrates. A recent review [3] describes several applications of this method.
The DNA-binding domains of E. coli uracil DNA-glycosilase (Ung) and of human Replication Protein A (RPA) were investigated using this methodology.
Ung catalyzes the cleavage of the N-glycosylic bond that joins the uracil base to the deoxyribose phosphate backbone of DNA. The enzyme recognizes uracil residues present in DNA as a result of dUMP incorporation or of deamination of cytosine and plays an important role in initiating the uracil-DNA excision repair pathway. In the case of Ung, several aminoacids (Tyr66, His67, His187, Pro188/Ser189, and His194) were putatively identified as sites that photocross-link to oligonucleotide dT20.
RPA is a single-standed DNA-binding protein that is highly conserved in eukaryotic cells. RPA was originally identified as a factor essential for the replication of simian virus SV40 DNA in vitro and subsequently has also been shown to play an important role in DNA repair and recombination. Using the same purification protocol, three aromatic amino acids (F238, F269 and F386) from RPA were identified as being photocross-linked to oligonucleotide dT30.
These observations are in good agreement with previously published data regarding the DNA binding domains of Ung and RPA obtained from crystal structure [5,6] and from site-directed mutagenesis experiments [7,8]. They illustrate that photochemical cross-linking and mass spectrometry can be successfully employed in the study of non-covalent nucleoprotein complexes. In addition, these experimental results demonstrate that mass spectrometry is an extremely versatile and powerful tool in biology.
References:
1. Jensen, O. N., Barofsky, D.F., Young, M.C., Von Hippel, P.H., Swenson, S., and Seifried, S (1993) "Direct observation of UV-cross-linked protein-nucleic acid complexes by matrix-assisted laser desorption ionization mass spectrometry" Rapid Commun. Mass Spectrom. 7, 496-501.
2. Veenstra, T. (1999) "Electrospray ionization mass spectrometry: a promising new technique in the study of protein/DNA noncovalent complexes" Biochem. Biophys. Res. Commun. 257, 1-5.
3. Steen, H. and Jensen, O.N. (2002) "Analysis of protein-nucleic acid interactions by photochemical cross-linking and mass spectrometry" Mass Spec. Rev. 21, 163-182.
4. Bennett, S. E., Jensen, O.N., Barofsky, D.F., and Mosbaugh, D.W. (1994) "UV-catalyzed cross-linking of Escherichia coli uracil-DNA glycosylase to DNA. Identification of amino acid residues in the single-stranded DNA binding site" J. Biol. Chem. 269, 21870-21879.
5. Xiao, G., Tordova, M., Jagadeesh, J., Drohat, A. C., Stivers, J. T. and Gilliland, G. L. (1999) "Crystal structure of Escherichia coli uracil DNA glycosylase and its complexes with uracil and glycerol: structure and glycosylase mechanism revisited" Proteins: Struct.,Funct., Genet. 35, 13-20.
6. Bochkarev, A., Pfuetzner, R.A., Edwards, A.M., and Frappier, L. (1997) "Structure of the single-stranded-DNA-binding domain of replication protein A bound to DNA" Nature 385, 176-181.
7. Shroyer, M.J., Bennett, S.E., Putnam, C.D., Tainer, J.A. and Mosbaugh, D.W. (1999) "Mutation of an active site residue in Escherichia coli uracil-DNA glycosylase: effect on DNA binding, uracil inhibition and catalysis" Biochemistry 39, 4834-4845.
8. Walther, A. P., Gomes X.V., Lao Y., Lee C.G., and Wold, M.S. (1999) "Replication protein A interactions with DNA.1. Functions of the DNA-binding and zinc-finger domains of the 70-kDa subunit" Biochemistry 38, 3963-3973.