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J Biol Chem ; 281(33): 23852-69, 2006 Aug 18.
Article in English | MEDLINE | ID: mdl-16675462

ABSTRACT

The functional and structural consequences of a mutation of the DNA intercalating residue of HincII, Q138F, are presented. Modeling has suggested that the DNA intercalation by Gln-138 results in DNA distortions potentially used by HincII in indirect readout of its cognate DNA, GTYRAC (Y = C or T, R = A or G) (Horton, N. C., Dorner, L. F., and Perona, J. J. (2002) Nat. Struct. Biol. 9, 42-47). Kinetic data presented here indicate that the mutation of glutamine 138 to phenylalanine (Q138F) results in a change in sequence specificity at the center two base pairs of the cognate recognition site. We show that the preference of HincII for cutting, but not binding, the three cognate sites differing in the center two base pairs has been altered by the mutation Q138F. Five new crystal structures are presented including Q138F HincII bound to GTTAAC and GTCGAC both with and without Ca2+ as well as the structure of wild type HincII bound to GTTAAC. The Q138F HincII/DNA structures show conformational changes in the protein, bound DNA, and at the protein-DNA interface, consistent with the formation of adaptive complexes. Analysis of these structures and the effect of Ca2+ binding on the protein-DNA interface illuminates the origin of the altered specificity by the mutation Q138F in the HincII enzyme.


Subject(s)
Bacterial Proteins/chemistry , Bacterial Proteins/metabolism , Base Sequence , Deoxyribonucleases, Type II Site-Specific/chemistry , Deoxyribonucleases, Type II Site-Specific/metabolism , Amino Acid Substitution/genetics , Bacterial Proteins/genetics , Calcium/metabolism , Crystallography, X-Ray , DNA, Bacterial/chemistry , DNA, Bacterial/genetics , DNA, Bacterial/metabolism , Deoxyribonucleases, Type II Site-Specific/genetics , Glutamine/genetics , Haemophilus influenzae/chemistry , Haemophilus influenzae/genetics , Haemophilus influenzae/metabolism , Hydrolysis , Intercalating Agents/chemistry , Intercalating Agents/metabolism , Kinetics , Mutagenesis, Site-Directed , Nucleic Acid Conformation , Phenylalanine/genetics , Protein Binding , Substrate Specificity/genetics
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