The MLH1G67R mutation does not have a significant impact on SHM. (A) Alignment of the GxG motif of MLH1 ATPase domain and a 2.5-Å resolution of the backbone of hMLH1 ATP-binding pocket with ATP (Protein Data Bank accession no. 3NA3). G67 of human MLH1 is highlighted with a gray oval, and the ATP molecule is shown inside the MLH1 ATPase pocket. Asterisks signify that the amino acid conservation is identical, and colons signify similarity at the respective amino acid residues. On the right, a cartoon backbone and a surface representation of the first 347 aa residues of mammalian MLH1 along with an ATP molecule trapped in the ATPase domain are shown. The G67 residue does not seem to be in a region having secondary structures but is in close proximity to the adenine base of the ATP molecule and not to the triphosphate group. Although MLH1 was crystallized as a homodimer, for simplicity, only the monomer is shown here. (B) Global analysis of unique mutation frequencies corrected for base composition according to the SHMTool algorithm. (C) Distribution of mutations in the V_H186.2 sequence (273 bp). (D) The spectrum of base substitutions is expressed as frequencies of mutation (x10⁻² mutations/base) and corrected for base composition. The cumulative spectrum of mutations from G, C, A, or T sites is also shown (Sum). (E) Proportion of sequences with different load of mutations (indicated in each fraction) for antigen and nonantigen selected regions (V_H186.2 and Jh2-Jh4, respectively). The center of the pies represents the total number of sequences analyzed in each group. (F) Relative frequency of mutations at G:C versus A:T sites observed in V_H186.2 and Jh2-Jh4 regions.