title: 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.
authors.affiliation: Department of Biological Sciences and Chemistry, University of Southern California, Los Angeles, California 90089-2910, USA.
authors.forename: Phuong
authors.initials: P
authors.lastname: Pham
authors.forename: Ke
authors.initials: K
authors.lastname: Zhang
authors.forename: Myron F
authors.initials: MF
authors.lastname: Goodman
mesh_terms: D001402:B-Lymphocytes / Q000378:metabolism*; D001483:Base Sequence; D053843:DNA Mismatch Repair / Q000235:genetics*; D006801:Humans; D008969:Molecular Sequence Data; D009154:Mutation / Q000235:genetics
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