Probing the binding region of the single-stranded DNA-binding domain of rat DNA polymerase beta using nanosecond-pulse laser-induced cross-linking and mass spectrometry.
In recent years, there has been a significant number of studies in which UV light has been used as a reagent to induce cross-links in nucleic acid-protein complexes. An area of considerable interest among those interested in structural biology is the garnering of information about the sites of cross-linking within the protein and nucleic acid members of photolinked conjugates, under the assumption that such knowledge should lead to identification of contact regions or sites within the native complexes. In this paper, we present our results from a photocross-linking study of the complex of the single-stranded DNA-binding domain of rat DNA polymerase beta (pol beta-ss) with the oligonucleotide d(ATATATA). In this study, we have used single nanosecond laser pulses as the cross-linking reagent and matrix-assisted laser desorption/ionization-time of flight mass spectrometry as an analytical tool to identify cross-linked peptides purified from proteolytic digests of the cross-linked complex. Six cross-linked peptides have been identified in tryptic digests of the protein-oligonucleotide conjugates that result from irradiation of the pol beta-ss-d(ATATATA) complex with a single laser pulse. Comparisons with NMR data in the literature for the same complex show that each of the cross-linked peptides contains amino acids that are in contact with the nucleic acid component of the complex.
Connor
D A
DA
Department of Pharmaceutical Chemistry, University of California, San Francisco 94143-0446, USA.
Falick
A M
AM
Young
M C
MC
Shetlar
M D
MD
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D001665:Binding Sites; D019951:DNA Polymerase beta / Q000737:chemistry* / Q000378:metabolism; D004277:DNA, Single-Stranded / Q000737:chemistry* / Q000378:metabolism; D004268:DNA-Binding Proteins / Q000737:chemistry / Q000378:metabolism; D007834:Lasers; D008958:Models, Molecular; D008969:Molecular Sequence Data; D009690:Nucleic Acid Conformation; D009838:Oligodeoxyribonucleotides / Q000737:chemistry / Q000378:metabolism; D010446:Peptide Fragments / Q000737:chemistry; D011487:Protein Conformation; D051381:Rats; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization
D004277:DNA, Single-Stranded; D004268:DNA-Binding Proteins; D009838:Oligodeoxyribonucleotides; D010446:Peptide Fragments; D011994:Recombinant Proteins; D019951:DNA Polymerase beta