Figure 5.
Figure 5. Mutation spectra from in vitro transcription-coupled mutation assays. (A) The T7-lacZ transcription-coupled substrate in which a T7 promoter is inserted upstream of the lac promoter in M13mp19 (Fig. S5 B). Nucleotide position 1 is defined as the start of the lac promoter. The adjacent table shows the 5′-flanking nucleotide preferences of the C mutations produced by GST-AID1 or GST-AID1*/3G in lac target DNA during in vitro transcription by phage T7 RNA polymerase. (B) Mutation distribution along the T7 transcription-coupled lacZ target DNA with mutation at each nucleotide position expressed as the percentage of total mutations. Two heavily mutated positions are off-scale: their percentage mutations are indicated. Because mutation analysis was restricted to Lac− plaques, this selection results in a skewing in favor of lac-inactivating mutations, although most mutated templates carried 1–3 mutations in the target region. Positions at which C deamination yields a stop codon are indicated by an asterisk. (C) The location and local context of the five most frequently mutated C residues along the transcribed lac target. All the mutated residues shown are located on the top (nontranscribed) strand. (D) Transcription-linked mutation of a T7-linked GFP-Vλ target. The substrate DNA is a derivative of plasmid pCR-Blunt II-TOPO in which a region of IgVλ (residues 115–164 or 228–263) has been inserted between the T7 promoter and a GFP reporter with T7-catalyzed transcription of the IgVλ fragment being in the same sense as IgVλ transcription in B cells (Fig. S5 C). For each construct (pCR-GFP-Vλ115−164-T7 and pCR-GFP-Vλ228−263-T7), the tables compare the percentage of total mutations within the target Vλ region that occur at selected individual positions with the equivalent percentage mutation values for the same positions over the same target regions in the M13 or B cell mutation assays. (E) Comparison of the mutation distributions obtained in the T7-coupled mutation assay over IgVλ residues 115–164 (for AID1) or 228–263 (for AID1*/3G) to the distributions obtained in DT40 B cells (left) or in the gapped duplex assay (right). In these comparisons, analysis is restricted to nontranscribed strand mutations. Positions of individual hotspots are indicated in italics. Red, 5′-purine flank; blue, 5′-pyrimidine flank.

Mutation spectra from in vitro transcription-coupled mutation assays. (A) The T7-lacZ transcription-coupled substrate in which a T7 promoter is inserted upstream of the lac promoter in M13mp19 (Fig. S5 B). Nucleotide position 1 is defined as the start of the lac promoter. The adjacent table shows the 5′-flanking nucleotide preferences of the C mutations produced by GST-AID1 or GST-AID1*/3G in lac target DNA during in vitro transcription by phage T7 RNA polymerase. (B) Mutation distribution along the T7 transcription-coupled lacZ target DNA with mutation at each nucleotide position expressed as the percentage of total mutations. Two heavily mutated positions are off-scale: their percentage mutations are indicated. Because mutation analysis was restricted to Lac− plaques, this selection results in a skewing in favor of lac-inactivating mutations, although most mutated templates carried 1–3 mutations in the target region. Positions at which C deamination yields a stop codon are indicated by an asterisk. (C) The location and local context of the five most frequently mutated C residues along the transcribed lac target. All the mutated residues shown are located on the top (nontranscribed) strand. (D) Transcription-linked mutation of a T7-linked GFP-Vλ target. The substrate DNA is a derivative of plasmid pCR-Blunt II-TOPO in which a region of IgVλ (residues 115–164 or 228–263) has been inserted between the T7 promoter and a GFP reporter with T7-catalyzed transcription of the IgVλ fragment being in the same sense as IgVλ transcription in B cells (Fig. S5 C). For each construct (pCR-GFP-Vλ115−164-T7 and pCR-GFP-Vλ228−263-T7), the tables compare the percentage of total mutations within the target Vλ region that occur at selected individual positions with the equivalent percentage mutation values for the same positions over the same target regions in the M13 or B cell mutation assays. (E) Comparison of the mutation distributions obtained in the T7-coupled mutation assay over IgVλ residues 115–164 (for AID1) or 228–263 (for AID1*/3G) to the distributions obtained in DT40 B cells (left) or in the gapped duplex assay (right). In these comparisons, analysis is restricted to nontranscribed strand mutations. Positions of individual hotspots are indicated in italics. Red, 5′-purine flank; blue, 5′-pyrimidine flank.

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