Asymmetric photocross-linking pattern of restriction endonuclease EcoRII to the DNA recognition sequence.
The EcoRII homodimer engages two of its recognition sequences (5'-CCWGG) simultaneously and is therefore a type IIE restriction endonuclease. To identify the amino acids of EcoRII that interact specifically with the recognition sequence, we photocross-linked EcoRII with oligonucleotide substrates that contained only one recognition sequence for EcoRII. In this recognition sequence, we substituted either 5-iododeoxycytidine for each C or 5-iododeoxyuridine for A, G, or T. These iodo-pyrimidine bases were excited using a UV laser to result in covalent cross-linking products. The yield of EcoRII photocross-linked to the 5'-C of the 5'-CCAGG strand of the recognition sequence was 45%. However, we could not photocross-link EcoRII to the 5'-C of the 5'-CCTGG strand. Thus, the contact of EcoRII to the bases of the recognition sequence appears to be asymmetric, unlike that expected for most type II restriction endonucleases. Tryptic digestion of free and of cross-linked EcoRII, followed by high performance liquid chromatography (HPLC) separation of the individual peptides and Edman degradation, identified amino acids 25-49 of EcoRII as the cross-linking peptide. Mutational analysis of the electron-rich amino acids His(36) and Tyr(41) of this peptide indicates that Tyr(41) is the amino acid involved in the cross-link and that it therefore contributes to specific DNA recognition by EcoRII.
Mücke
Merlind
M
Institut für Virologie, Medizinische Fakultät der Humboldt-Universität zu Berlin (Charité), D-10098 Berlin, Germany.
Pingoud
Vera
V
Grelle
Gerlinde
G
Kraft
Regine
R
Krüger
Detlev H
DH
Reuter
Monika
M
D001483:Base Sequence; D002851:Chromatography, High Pressure Liquid; D003432:Cross-Linking Reagents / Q000494:pharmacology*; D004247:DNA / Q000378:metabolism*; D004252:DNA Mutational Analysis; D015252:Deoxyribonucleases, Type II Site-Specific / Q000737:chemistry* / Q000378:metabolism*; D004591:Electrophoresis, Polyacrylamide Gel; D006639:Histidine / Q000737:chemistry; D008969:Molecular Sequence Data; D009154:Mutation; D009841:Oligonucleotides / Q000737:chemistry; D010455:Peptides / Q000737:chemistry; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D013997:Time Factors; D014364:Tryptophan / Q000737:chemistry
D003432:Cross-Linking Reagents; D009841:Oligonucleotides; D010455:Peptides; D006639:Histidine; D014364:Tryptophan; D004247:DNA; C046545:CCWGG-specific type II deoxyribonucleases; D015252:Deoxyribonucleases, Type II Site-Specific