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Integrated mimicry of B cell antibody mutagenesis using yeast homologous recombination.

ABSTRACT: Antibody affinity maturation proceeds in vivo via a combination of point mutations, insertions, deletions, and combinatorial shuffling of light chains or portions of the heavy chain, thereby reducing the probability of trapping in local affinity optima in sequence space. In vivo homologous recombination in yeast can be exploited to mimic the broad spectrum of mutational types deployed by B cells, incorporating both receptor revision and receptor editing together with polymerase-directed point mutagenesis. This method was used to effect a 10,000-fold affinity improvement in an anti-peptide single-chain antibody in three rounds of mutagenesis and screening, and a 1,000-fold affinity improvement in an anti-protein single-chain antibody in a single round. When recombinational mutagenesis (CDR or chain shuffling) was directly compared to error-prone PCR, the recombinational approach yielded greater affinity improvement with substantially reduced divergence from germline sequences, demonstrating an advantage of simultaneously testing a broad range of mutational strategies.

SUBMITTER: Swers JS

PROVIDER: S-EPMC3986863 | biostudies-literature | 2011 Jan

REPOSITORIES: biostudies-literature

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Integrated mimicry of B cell antibody mutagenesis using yeast homologous recombination.

Swers Jeffrey S JS Yeung Yik A YA Wittrup K Dane KD

Molecular biotechnology 20110101 1

Antibody affinity maturation proceeds in vivo via a combination of point mutations, insertions, deletions, and combinatorial shuffling of light chains or portions of the heavy chain, thereby reducing the probability of trapping in local affinity optima in sequence space. In vivo homologous recombination in yeast can be exploited to mimic the broad spectrum of mutational types deployed by B cells, incorporating both receptor revision and receptor editing together with polymerase-directed point mu ...[more]

PMID: 20645027

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Project description:The Saccharomyces cerevisae RAD3 gene is the homolog of human XPD, an essential gene encoding a DNA helicase of the TFIIH complex involved in both nucleotide excision repair (NER) and transcription. Some mutant alleles of RAD3 (rad3-101 and rad3-102) have partial defects in DNA repair and a strong hyper-recombination (hyper-Rec) phenotype. Previous studies showed that the hyper-Rec phenotype associated with rad3-101 and rad3-102 can be explained as a consequence of persistent single-stranded DNA gaps that are converted to recombinogenic double-strand breaks (DSBs) by replication. The systems previously used to characterize the hyper-Rec phenotype of rad3 strains do not detect the reciprocal products of mitotic recombination. We have further characterized these events using a system in which the reciprocal products of mitotic recombination are recovered. Both rad3-101 and rad3-102 elevate the frequency of reciprocal crossovers about 100-fold. Mapping of these events shows that three-quarters of these crossovers reflect DSBs formed at the same positions in both sister chromatids (double sister-chromatid breaks, DSCBs). The remainder reflects DSBs formed in single chromatids (single chromatid breaks, SCBs). The ratio of DSCBs to SCBs is similar to that observed for spontaneous recombination events in wild-type cells. We mapped 216 unselected genomic alterations throughout the genome including crossovers, gene conversions, deletions, and duplications. We found a significant association between the location of these recombination events and regions with elevated gamma-H2AX. In addition, there was a hotspot for deletions and duplications at the IMA2 and HXT11 genes near the left end of chromosome XV. A comparison of these data with our previous analysis of spontaneous mitotic recombination events suggests that a sub-set of spontaneous events in wild-type cells may be initiated by incomplete NER reactions, and that DSCBs, which cannot be repaired by sister-chromatid recombination, are a major source of mitotic recombination between homologous chromosomes.

Dataset Information

Integrated mimicry of B cell antibody mutagenesis using yeast homologous recombination.

Publications

Integrated mimicry of B cell antibody mutagenesis using yeast homologous recombination.

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