Protein Engineering Protocols - Mycobacteriology research center

116 Friedhoff and PingoudFig. 2. Superposition of MutH with restriction endonucleases. (A) The two nucleotidesof the MunI recognition sequence corresponding to the two adenines of theMutH recognition sequence, d(GATC), are shown. The nucleotide with the asterisks(A*) corresponds to the adenine of the MutH recognition sequence in the lower strand,which can be methylated and then directs cleavage to the upper strand. (B,C)Quantitative analysis of the superposition of MutH with 11 different restriction endonucleases(BamHI, BglI, BglII, BsoBI, EcoRI, EcoRV, HincII, MunI, NaeI, NgoMIV,and PvuII). Shown are the average distances of the atoms of the MutH structure to theatom N6 (adenine), O6 (guanine), N4 (cytosine), or O4 (thymine) of the nucleotidecorresponding in position to the adenine in the upper strand (B) or lower strand (C),respectively, of the MutH recognition sequence. Note the minima, which indicate theproximity of residues, i.e., K48, F94, R184, and Y212, to the N6 of adenine or itsequivalent in both the upper and lower strand, respectively, in all of the superimposedDNA structures.3. GeneDoc (http://www.psc.edu/biomed/genedoc/).4. RasMol (http://www.openrasmol.org/).5. Swiss PDB Viewer (http://www.expasy.org/spdbv/).2.2. Site-Directed Mutagenesis of MutH1. pMQ402, a BAD18 derivative, is used as a bacterial expression plasmid (23).Under control of an arabinose promoter for inducible protein expression, it containsthe coding sequence for a His-tagged MutH protein, which can be purifiedby Ni-nitrilotriacetic acid (NTA) agarose affinity chromatography. The plasmidharbors an ampicillin-resistance gene for selection.