Identification of a base-specific contact between the restriction endonuclease SsoII and its recognition sequence by photocross-linking
AUTOR(ES)
Kubareva, Elena A.
FONTE
Oxford University Press
RESUMO
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.
ACESSO AO ARTIGO
http://www.pubmedcentral.nih.gov/articlerender.fcgi?artid=102617Documentos Relacionados
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