Different IgVλ hotspots dominate the mutation spectra in B cells and in the gapped duplex assay. (A) The M13mp19-IgVλ-lacZ gapped duplex substrate DNA is depicted with more detailed information provided in Fig. S5 A. Nucleotide position 1 is equivalent to the first nucleotide of the in vivo IgVλ analyzed in Fig. 3. (B) The graphs compare the distribution of IgVλ mutations obtained with AID¹ or AID¹*/3G in the gapped duplex assay (shown below the line) with the distribution of IgVλ top strand C mutations obtained with the same deaminases in DT40 B cells (shown above the line). The distributions of IgVλ top strand C mutations in DT40 cells for AID¹*/3G and AID¹ derive from the same mutation databases used in Fig. 3 and Fig. S2, respectively, although those figures portray all C mutations (i.e., whichever DNA strand they have occurred upon). The positions of some individual hotspots are indicated in italics. Red, 5′-purine flank (Pu-C); blue, 5′-pyrimidine flank (Py-C). (C) The location and local context of the most frequently mutated C residues along the IgVλ top strand in the gapped duplex and DT40 B cell mutation assays are compared. Mutation load at each nucleotide position is represented as a percentage of the total mutations in each dataset. (D) The percentages of total C mutations in the gapped duplex assays and DT40 B cells (top strand) at C residues with each of the four possible 5′-flanking bases are compared for AID¹ and AID¹*/3G.