Identification of a base-specific contact between the restriction endonuclease SsoII and its recognition sequence by photocross-linking.
A target sequence-specific DNA binding region of the restriction endonuclease Sso II was identified by photocross-linking with an oligodeoxynucleotide duplex which was substituted with 5-iododeoxy-uridine (5-IdU) at the central position of the Sso II recognition site (CCNGG). For this purpose the Sso II-DNA complex was irradiated with a helium/cadmium laser (325 nm). The cross-linking yield obtained was approximately 50%. In the presence of excess unmodified oligodeoxynucleotide or with oligode-oxynucleotides substituted with 5-IdU elsewhere, no cross-linking was observed, indicating the specificity of the cross-linking reaction. The cross-linked Sso II-oligodeoxynucleotide complex was digested with chymotrypsin, a cross-linked peptide-oligodeoxy-nucleotide 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 Sso II to its DNA substrate.
Kubareva
E A
EA
A. N. Belozersky Institute of Physical and Chemical Biology and Chemistry Department, Moscow State University, Moscow 119899, Russia.
Thole
H
H
Karyagina
A S
AS
Oretskaya
T S
TS
Pingoud
A
A
Pingoud
V
V
D020029:Base Pairing; D001483:Base Sequence; D001665:Binding Sites; D004247:DNA / Q000235:genetics* / Q000378:metabolism; D015252:Deoxyribonucleases, Type II Site-Specific / Q000235:genetics* / Q000378:metabolism; D004926:Escherichia coli; D008969:Molecular Sequence Data; D011485:Protein Binding
D004247:DNA; D015252:Deoxyribonucleases, Type II Site-Specific