Studies of the DNA mismatch binding proteins MutS, VSR and CEL I

Abstract

One of the aims of this work was to probe MutS by site directed mutagenesis to identify amino acids that are important in mismatch recognition.  Several site-directed mutants of MutS were expressed and purified and bandshift assays were done to assess their affinity for different DNA mismatches.  F36 and M68 were found to be essential for mismatch binding, while E38 was found to be important but not essential.  E38 was particularly important for binding to GT and GG mismatches, since the affinity for these mismatches was lost when E38 was mutated to A.  Mutating E38Q caused the protein to lose specificity for mismatches and to bind mismatches and Watson-Crick DNA with similar affinities.  The mutation L39C, Y41G also resulted in the loss of the high affinity binding to G.T. and G.G. mismatches but the binding affinity for most other mismatches was not affected.

VSR endonuclease cleaves T.G. mismatches within sequence CT(A/T)GG, where the underlined T is paired with a G.  Mismatch recognition by this enzyme was probed by site directed mutagenesis.  A truncated mutant was created that was found to cleave the substrate, but at 100 times slower rate than the wild-type protein.  Substrate binding by this mutant was not captured in a gel shift assay.  D51 was found to be essential for substrate cleavage by VSR, but not absolutely essential for substrate binding since the mutant D51A is still able to bind to substrate DNA, albeit with a lower affinity.  F67, W68 and H69 appear to be absolutely essential for both substrate binding and for hydrolysis by VSR.

CEL I nuclease was originally isolated from celery stalks and is a eukaryotic protein that could be used to detect DNA mismatches.  The recombinant CEL I protein was found to cleave DNA in a mismatch specific manner but background cleavage of homoduplex DNA was also evident.

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