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
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