Nucleic Acids Research, 2000, Vol. 28, No. 5 1085-1091
© 2000 Oxford University Press
Identification of a base-specific contact between the restriction endonuclease SsoII and its recognition sequence by photocross-linking
A. N. Belozersky Institute of Physical and Chemical Biology and Chemistry Department, Moscow State University, Moscow 119899, Russia, 1Zentrum Kinderheilkunde, Medizinische Hochschule Hannover, Carl-Neuberg-Straße 1, D-30623 Hannover, Germany, 2Institute of Biomedical Chemistry, Russian Academy of Medical Sciences, Pogodinskaya str. 10, Moscow 119121, Russia and 3Institut für Biochemie, Fachbereich 08, Justus-Liebig-Universität, Heinrich-Buff-Ring 58, D-35392 Giessen, Germany
A target sequence-specific DNA binding region of the restriction endonuclease SsoII was identified by photocross-linking with an oligodeoxynucleotide duplex which was substituted with 5-iododeoxyuridine (5-IdU) at the central position of the SsoII recognition site (CCNGG). For this purpose the SsoII–DNA complex was irradiated with a helium/cadmium laser (325 nm). The cross-linking yield obtained was ~50%. In the presence of excess unmodified oligodeoxynucleotide or with oligodeoxynucleotides substituted with 5-IdU elsewhere, no cross-linking was observed, indicating the specificity of the cross-linking reaction. The cross-linked SsoII-oligodeoxynucleotide complex was digested with chymotrypsin, a cross-linked peptide–oligodeoxynucleotide complex isolated and the site of cross-linking identified by Edman sequencing to be Trp61. In line with this identification is the finding that the W61A variant cannot be cross-linked with the IdU-substituted oligodeoxynucleotide, shows a decrease in affinity towards DNA and is inactive in cleavage. It is concluded that the region around Trp61 is involved in specific binding of SsoII to its DNA substrate.
* To whom correspondence should be addressed. Tel: +49 641 99 35402; Fax: +49 641 99 35409; Email: vera.pingoud@chemie.bio.uni-giessen.de This paper is dedicated to the memory of Professor Dr Z. Shabarova (
19 September 1999) and Professor Dr D. Nathans ( 16 November 1999), pioneers in the study of the biochemistry of restriction enzymes
CiteULike 
Connotea 
Del.icio.us What's this?
This article has been cited by other articles:
|
|
 |
|
  H. Geyer, R. Geyer, and V. Pingoud A novel strategy for the identification of protein-DNA contacts by photocrosslinking and mass spectrometry Nucleic Acids Res., September 21, 2004; 32(16): e132 - e132. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 M. Mucke, V. Pingoud, G. Grelle, R. Kraft, D. H. Kruger, and M. Reuter Asymmetric Photocross-linking Pattern of Restriction Endonuclease EcoRII to the DNA Recognition Sequence J. Biol. Chem., April 12, 2002; 277(16): 14288 - 14293. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
V. Pingoud, E. Kubareva, G. Stengel, P. Friedhoff, J. M. Bujnicki, C. Urbanke, A. Sudina, and A. Pingoud Evolutionary Relationship between Different Subgroups of Restriction Endonucleases J. Biol. Chem., April 12, 2002; 277(16): 14306 - 14314. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 H. Steen, J. Petersen, M. Mann, and O. N. Jensen Mass spectrometric analysis of a UV-cross-linked protein-DNA complex: Tryptophans 54 and 88 of E. coli SSB cross-link to DNA Protein Sci., October 19, 2001; 10(10): 1989 - 2001. [Abstract] [Full Text] [PDF]
|
|