Mutagenesis dependent upon the combination of activation-induced deaminase expression and a double-strand break

Laboratory of Structural Biology, National Institutes of Health, NIEHS, 111 T.W. Alexander Drive, PO Box 12233, MD F1-12, Research Triangle Park, NC 27709, USA.
Molecular Immunology (Impact Factor: 2.97). 11/2010; 48(1-3):164-70. DOI: 10.1016/j.molimm.2010.08.013
Source: PubMed


We explored DNA metabolic events potentially relevant to somatic hypermutation (SHM) of immunoglobulin genes using a yeast model system. Double-strand break (DSB) formation has been discussed as a possible component of the SHM process during immunoglobulin gene maturation. Yet, possible mechanisms linking DSB formation with mutagenesis have not been well understood. In the present study, a linkage between mutagenesis in a reporter gene and a double-strand break at a distal site was examined as a function of activation-induced deaminase (AID) expression. Induction of the DSB was found to be associated with mutagenesis in a genomic marker gene located 7 kb upstream of the break site: mutagenesis was strongest with the combination of AID expression and DSB induction. The mutation spectrum of this DSB and AID-mediated mutagenesis was characteristic of replicative bypass of uracil in one strand and was dependent on expression of DNA polymerase delta (Polδ). These results in a yeast model system illustrate that the combination of DSB induction and AID expression could be associated with mutagenesis observed in SHM. Implications of these findings for SHM of immunoglobulin genes in human B cells are discussed.

Download full-text


Available from: Rajendra Prasad,
  • Source
    • "We chose to use APOBEC3G* for this experiment since it gave a good mutation load but a lower proportion of kataegic mutations than AID* (Supplementary file 1B): any enhancement of kataegis would therefore be more readily detectable. Consistent with previous findings (Poltoratsky et al., 2010), induction of I-SceI expression resulted in an increased frequency canonical APOBEC3G CC context. Two-thirds of the CAN1 region mutations in the KanMX-ISceI RS cells were transversions. "
    [Show abstract] [Hide abstract]
    ABSTRACT: eLife digest The genomes of cancer cells contain mutations that are not present in normal cells. Some of these prevent cells from repairing their DNA, while others give rise to tumours by causing cells to multiply uncontrollably. Moreover, some of the mutations in breast cancer cells occur in clusters—a phenomenon known as kataegis (from the Greek for ‘thunderstorm’). Kataegic mutations occur almost exclusively at a cytosine preceded by a thymine. This suggests that a family of proteins called AID/APOBEC enzymes—which remove amine groups from cytosines—may be involved in generating these mutations. In this study, Taylor et al. confirm this possibility by showing that expressing individual members of the AID/APOBEC family of enzymes in yeast cells increases the mutation frequency and induces kataegis. The kataegis triggered by the AID/APOBEC enzymes could be localised through the introduction of double-stranded breaks into the DNA: Taylor et al. suggest that this might happen because repairing the breaks exposes single-stranded DNA, which the AID/APOBEC enzymes then act upon. By comparing the mutations induced in the yeast cells with those observed in breast cancer cells, Taylor et al. identified APOBEC3B as the enzyme most likely to be responsible for kataegis in breast cancer (with APOBEC3A also a strong candidate in some cancers). Moreover, they showed that APOBEC3B was highly expressed in breast cancer cell lines, and that APOBEC3B and APOBEC3A can also cause DNA damage in human cells. Taken together, the findings provide key insights into the mechanism by which kataegis arises, and identify two proteins likely to contribute to the mutations seen in breast cancer. Further work is now required to determine whether these enzymes also give rise to mutations in other forms of cancer. DOI:
    eLife Sciences 04/2013; 2(2):e00534. DOI:10.7554/eLife.00534 · 9.32 Impact Factor
  • Source
    • "Regions of ssDNA are recognized as a prerequisite for kataegis-like events induced by an alkylation agent in yeast, and by extrapolation, have been proposed to be a prerequisite for the kataegistic action of deaminases in humans [3]. Double-strand DNA breaks in the vicinity of a reporter gene synergistically stimulate mutagenesis by AID, and in yeast this behavior might be related to the generation of ssDNA during homologous recombination [8]. "
    [Show abstract] [Hide abstract]
    ABSTRACT: Clusters of localized hypermutation in human breast cancer genomes, named “kataegis” (from the Greek for thunderstorm), are hypothesized to result from multiple cytosine deaminations catalyzed by AID/APOBEC proteins. However, a direct link between APOBECs and kataegis is still lacking. We have sequenced the genomes of yeast mutants induced in diploids by expression of the gene for PmCDA1, a hypermutagenic deaminase from sea lamprey. Analysis of the distribution of 5,138 induced mutations revealed localized clusters very similar to those found in tumors. Our data provide evidence that unleashed cytosine deaminase activity is an evolutionary conserved, prominent source of genome-wide kataegis events. Reviewers This article was reviewed by: Professor Sandor Pongor, Professor Shamil R. Sunyaev, and Dr Vladimir Kuznetsov.
    Biology Direct 12/2012; 7(1):47. DOI:10.1186/1745-6150-7-47 · 4.66 Impact Factor
  • Source
    [Show abstract] [Hide abstract]
    ABSTRACT: Genetic information should be accurately transmitted from cell to cell; conversely, the adaptation in evolution and disease is fueled by mutations. In the case of cancer development, multiple genetic changes happen in somatic diploid cells. Most classic studies of the molecular mechanisms of mutagenesis have been performed in haploids. We demonstrate that the parameters of the mutation process are different in diploid cell populations. The genomes of drug-resistant mutants induced in yeast diploids by base analog 6-hydroxylaminopurine (HAP) or AID/APOBEC cytosine deaminase PmCDA1 from lamprey carried a stunning load of thousands of unselected mutations. Haploid mutants contained almost an order of magnitude fewer mutations. To explain this, we propose that the distribution of induced mutation rates in the cell population is uneven. The mutants in diploids with coincidental mutations in the two copies of the reporter gene arise from a fraction of cells that are transiently hypersensitive to the mutagenic action of a given mutagen. The progeny of such cells were never recovered in haploids due to the lethality caused by the inactivation of single-copy essential genes in cells with too many induced mutations. In diploid cells, the progeny of hypersensitive cells survived, but their genomes were saturated by heterozygous mutations. The reason for the hypermutability of cells could be transient faults of the mutation prevention pathways, like sanitization of nucleotide pools for HAP or an elevated expression of the PmCDA1 gene or the temporary inability of the destruction of the deaminase. The hypothesis on spikes of mutability may explain the sudden acquisition of multiple mutational changes during evolution and carcinogenesis.
    PLoS Genetics 09/2013; 9(9):e1003736. DOI:10.1371/journal.pgen.1003736 · 7.53 Impact Factor
Show more