Mass spectrometry of UV-cross-linked protein-nucleic acid complexes: identification of amino acid residues in the single-stranded DNA-binding domain of human replication protein A.

Photochemical cross-linking of human replication protein A (hRPA) to oligonucleotide dT30 was performed to enable identification of amino acid sequences that reside in the DNA-binding domain. A nucleoprotein complex, with a 1:1 protein/DNA stoichiometry, was separated from unreacted enzyme and oligonucleotide by SDS-polyacrylamide gel electrophoresis and subjected to in-gel digestion with trypsin. Three cross-linked tryptic peptides (nucleopeptides) of hRPA70xdT30 (T43, T28/29, and a truncated T24/25) were isolated. Combined mass spectrometric and C-terminal proteolysis experiments showed that at least one amino acid in the segment 235-ATAFNE-240 (located in T24/25), at least one out of the two residues sequence 269-FT-270 (located in T28/29), and at least one from the sequence 383-VSDF-386 (located in T43) were involved in cross-linking. These peptides contained aromatic residues (F238, F269, and F386 respectively) that can form base-stacking interactions with the DNA and were, therefore, most likely to be involved in cross-linking. The results obtained in this study demonstrate that a combination of exhaustive proteolysis and MALDI TOF MS can localize the sites of DNA binding to very short sequences of amino acids. Data so acquired can confirm or amend information obtained from site-directed mutagenesis and X-ray crystallography.