Hypermutation at A/T sites during G.U mismatch repair in vitro by human B-cell lysates

Abstract

Somatic hypermutation in the variable regions of immunoglobulin genes is required to produce high affinity antibody molecules. Somatic hypermutation results by processing G.U mismatches generated when activation-induced cytidine deaminase (AID) deaminates C to U. Mutations at C/G sites are targeted mainly at deamination sites, whereas mutations at A/T sites entail error-prone DNA gap repair. We used B-cell lysates to analyze salient features of somatic hypermutation with in vitro mutational assays. Tonsil and hypermutating Ramos B-cells convert C-->U in accord with AID motif specificities, whereas HeLa cells do not. Using tonsil cell lysates to repair a G.U mismatch, A/T and G/C targeted mutations occur about equally, whereas Ramos cell lysates make fewer mutations at A/T sites (approximately 24%) compared with G/C sites (approximately 76%). In contrast, mutations in HeLa cell lysates occur almost exclusively at G/C sites (> 95%). By recapitulating two basic features of B-cell-specific somatic hypermutation, G/C mutations targeted to AID hot spot motifs and elevated A/T mutations dependent on error-prone processing of G.U mispairs, these cell free assays provide a practical method to reconstitute error-prone mismatch repair using purified B-cell proteins.

FIGURE 1.

Schematic representation for an in vitro assay to measure C→U deamination activity and motif specificity in cell lysates. A circular M13mp2 gapped DNA substrate containing a 365-nt single-stranded DNA region, with a lacZα mutational reporter gene, is incubated with a cell lysate in the presence of UGI. Deamination products are analyzed after transfecting the DNA into E. coli lacking UNG, followed by plating on an α-complementation strain of E. coli. Deaminations occurring on individual DNA substrates are detected as C→T mutations in the lacZα gene of mutant M13 phages (white or light blue plaques), see “Experimental Procedures.” Any DNA that undergoes deamination within wild type E. coli will be linearized by the combined action of endogenous UNG and apurinic/apyrimidinic endonuclease, and therefore cannot form phage plaques.

FIGURE 2.

Deamination profiles produced by lysates from tonsil and Ramos B-cells and HeLa non-B-cells. a, deamination profiles produced by nuclear (♦, top) and cytoplasmic lysates (▪, bottom) prepared from activated tonsil B-cells. b, deamination profiles produced by nuclear lysates prepared from Ramos B-cells (•, top) and HeLa non-B-cells (▴, bottom). Each symbol above or below the DNA denotes a single deamination event. Red ovals denote a WRC hot spot motif, highly specific for AID-catalyzed C deamination. C₁₀₈ is a hot spot for A3G-catalyzed C deamination but a cold spot for deamination by AID.

FIGURE 3.

An M13 plaque assay designed to measure the efficiency of MMR of a single G·U mismatch with B-cell lysates. a, M13 plaques in the absence of cell lysates. b, M13 plaques reflecting MMR of a G·U mismatch in the presence of tonsil B-cell lysates. G·U heteroduplex repair is carried out by B-cell lysates in the presence of UGI, followed by transfection into MMR and BER (ung^- mutS^-) competent cells. The G·U mismatch repair efficiency is measured as the reduction in the fraction of mixed plaques (M) relative to the total number of plaques. Arrows with W, B, or M indicate examples of pure white, pure blue, or mixed plaques, respectively.

FIGURE 4.

Schematic representation of a reversion assay designed to measure error-prone MMR accuracy and specificity with cell lysates. Schematic at top shows a DNA molecule containing a G·U heteroduplex with a nonsense codon (TGA) in the plus strand of the lacZα gene and an AUT in the minus strand located opposite TGA. Repair of G·U is carried out by incubation of the heteroduplex with cell lysates in the presence of UGI. Accurate MMR of either the plus strand or the minus strand results in a phage carrying a nonsense codon in the lacZα gene that results in a white plaque phenotype. Error-prone MMR of either strand converts the nonsense codons into missense codons, resulting in a blue plaque phenotype.