This study aims to establish and exploit a new method of determining the location of DNA binding sites in proteins at the level of individual amino acids. The method is potentially applicable to any protein that binds non-covalently to DNA. UV light can induce protein-DNA crosslinking in cells and protein that binds to DNA has a much greater tendency to become crosslinked than unbound protein. Specifically, that portion of the protein that is in contact with the DNA will be crosslinked. Thus, UV light can be used as a """"""""zero-length"""""""" crosslinking reagent. Knowledge of the contact regions between DNA and protein in DNA-protein complexes is an essential prerequisite for achieving an understanding of biological processes at the molecular level. Proteins bind to DNA for a variety of essential purposes, as well as being implicated in cancer, as for example, the gene products of tumor suppressor genes and oncogenes. Two types of processes relevant to cancer research are transcriptional regulation and DNA repair. The two mainobstacles to the study of these crosslinked DNA-protein systems have been the difficulty of analyzing for the modified amino acids in the protein, and the lack of knowledge about the stability of the crosslinks formed. In order to overcome these obstacles, we propose to use molecular weight determination and sequencing by tandem mass spectrometry to perform the necessary peptide analyses. Peptides to be analyzed will be produced from the protein-DNA complex by enzyme digestion of the protein and the attached DNA as necessary. Initially, we intend to determine the exact amino acid residues in the rat DNA polymerase b protein that cross-link to DNA when exposed to UV light. We will also prepare and characterize peptide-nucleobase, peptide-oligonucleotide, and protein-oligonucleotide crosslinked species. Our initial goals are: 1) To establish that UV photoinduced crosslinking occurs between the amino acid residues and the nucleic acid bases at specific binding site(s) in the polymerase b protein. 2) To isolate and analyze crosslinked peptides by means of mass spectrometry. Model peptides and oligonuleotides will be used in the initial stages of this work. 3) To determine the optimum experimental conditions and procedures for production, separation, and analysis of crosslinked proteins and to establish reasonable minimum sample requirements.
Showing the most recent 10 out of 630 publications
