No direct contacts are made between HincII and the Y3 base of the recognition sequence, G1T2Y3R4A5C6, and residue Asn 141 is the only side chain of HincII to make a direct contact to R4. As seen below from the structure of wild type HincII bound to GTCGAC, the NH2 moiety (blue) of Asn 141 hydrogen bonds to the N7 of G4, which can be formed with either purine, A or G. This residue was mutated to alanine and the resulting N141A HincII analyzed. While the DNA cleavage rate and DNA binding affinity were greatly reduced as a result of the mutation, the specificity was completely wild type, showing cleavage only at GTYRAC sequences. This indicates that some other means besides the direct contact by N141 is used by HincII to recognize the center YR sequence in GTYRAC.
Cleavage of plasmid DNA bearing 14 GTNNAC sites, but only 3 GTYRAC sites:
The structure of N141A HincII bound to GTCGAC shows the same distortion at the center YR step as that induced by wild type HincII. It also unexpectedly shows a loss of DNA intercalation by Gln 138, and loss of direct contacts to the outer G1C6 base pair bases.
One subunit in the N141A HincII dimer rotates as rigid body by about 11 degrees relative to the other, opening up the DNA binding site. The loss of two hydrogen bonds from residue 141 to the DNA likely weakens the contacts sufficiently to stabilize this conformation, which may normally occur in the wild type HincII en route to the final recognition complex.2. Experiments with Q138F HincII
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Department of Biochemistry and Molecular Biophysics
University of Arizona
January 11, 2008
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