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

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• 作者：Catalin E. Doneanu ; Philip R. Gafken ; Samuel E. Bennett ; and Douglas F. Barofsky
• 刊名：Analytical Chemistry
• 出版年：2004
• 出版时间：October 1, 2004
• 年：2004
• 卷：76
• 期：19
• 页码：5667 - 5676
• 全文大小：270K
• 年卷期：v.76,no.19(October 1, 2004)
• ISSN：1520-6882

文摘

Photochemical cross-linking of human replication proteinA (hRPA) to oligonucleotide dT₃₀ was performed to enableidentification of amino acid sequences that reside in theDNA-binding domain. A nucleoprotein complex, with a1:1 protein/DNA stoichiometry, was separated from unreacted enzyme and oligonucleotide by SDS-polyacrylamide gel electrophoresis and subjected to in-gel digestionwith trypsin. Three cross-linked tryptic peptides (nucleopeptides) of hRPA70×dT₃₀ (T₄₃, T_28/29, and a truncated*T_24/25) were isolated. Combined mass spectrometric andC-terminal proteolysis experiments showed that at leastone amino acid in the segment 235-ATAFNE-240 (locatedin *T_24/25), at least one out of the two residues sequence269-FT-270 (located in T_28/29), and at least one from thesequence 383-VSDF-386 (located in T₄₃) were involvedin cross-linking. These peptides contained aromatic residues (F238, F269, and F386 respectively) that can formbase-stacking interactions with the DNA and were, therefore, most likely to be involved in cross-linking. Theresults obtained in this study demonstrate that a combination of exhaustive proteolysis and MALDI TOF MS canlocalize the sites of DNA binding to very short sequencesof amino acids. Data so acquired can confirm or amendinformation obtained from site-directed mutagenesis andX-ray crystallography.