Identification of base-specific contacts in protein-DNA complexes by photocrosslinking and mass spectrometry: a case study using the restriction endonuclease SsoII.
Specific protein-nucleic acid interactions are of paramount importance for the propagation, maintenance and expression of genetic information. Restriction endonucleases serve as model systems to study the mechanisms of DNA recognition by proteins. SsoII is a Type II restriction endonuclease that recognizes the double stranded sequence downward arrow CCNGG and cleaves it in the presence of Mg(2+)-ions, as indicated. SsoII shows sequence similarity over a stretch of approximately 70 amino acid residues with several other restriction endonucleases that recognize a similar sequence as SsoII (Cfr10I, EcoRII, NgoMIV, PspGI). In NgoMIV this stretch is involved in DNA recognition and cleavage, as shown by the crystal structure analysis of an enzyme-product complex. To find out whether the presumptive DNA recognition region in SsoII is indeed in contact with DNA we have photocrosslinked SsoII with an oligodeoxyribonucleotide in which the first guanine of the recognition sequence was replaced by 5-iodouracil. Following digestion by trypsin, the peptide-oligodeoxyribonucleotide conjugate was purified by Fe(3+)-IMAC and then incubated with hydrogen fluoride, which hydrolyzes the oligodeoxyribonucleotide to yield the peptide-deoxyuridine conjugate. The site of photocrosslinking was identified by MALDI-TOF-MS and MALDI-TOF-MS/MS to be Trp189, adjacent to Arg188, which aligns with Arg194 in NgoMIV, involved in recognition of the second guanine in the NgoMIV recognition sequence G downward arrow CCGGC. This result confirms previously published conclusions drawn on the basis of a mutational analysis of SsoII. The methodology that was employed here can be used in principle to identify the DNA binding site of any protein.
Pingoud
Vera
V
Institut für Biochemie (FB 08), Justus-Liebig-Universität, Heinrich-Buff-Ring 58, D-35392 Giessen, Germany. vera.pingoud@chemie.bio.uni-giessen.de
Geyer
Hildegard
H
Geyer
Rudolf
R
Kubareva
Elena
E
Bujnicki
Janusz M
JM
Pingoud
Alfred
A
D000595:Amino Acid Sequence; D001483:Base Sequence; D001665:Binding Sites; D003432:Cross-Linking Reagents / Q000737:chemistry*; D004247:DNA / Q000737:chemistry / Q000378:metabolism*; D015252:Deoxyribonucleases, Type II Site-Specific / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D008958:Models, Molecular; D008969:Molecular Sequence Data; D009838:Oligodeoxyribonucleotides / Q000737:chemistry / Q000378:metabolism; D011485:Protein Binding; D017386:Sequence Homology, Amino Acid; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization / Q000379:methods
D003432:Cross-Linking Reagents; D009838:Oligodeoxyribonucleotides; D004247:DNA; C045297:CCNGG-specific type II deoxyribonucleases; D015252:Deoxyribonucleases, Type II Site-Specific