title: Mutagenesis by AID, a molecule critical to immunoglobulin hypermutation, is not caused by an alteration of the precursor nucleotide pool.
abstract: The novel cytidine deaminase, AID, plays a critical role in immunoglobulin (Ig) hypermutation. Its possible modes of action include deamination of an RNA transcript that encodes a molecule involved in these processes, deamination of the DNA encoding the variable regions of immunoglobulin genes, or deamination of monomeric cytidine or deoxycytidine (dC) nucleotide generating a mutagenic imbalanced nucleotide pool. We transformed AID into Escherichia coli cells and measured the nucleotide pools at 2 and 6h following induction of expression. Although the majority of the cells expressed AID at the relevant time points, the nucleotide pools were unaltered. In addition, mutagenesis by AID expression in E. coli was not synergistically enhanced in a bacterial strain defective in dUTPase, an enzyme that prevents accumulation of dUTP in the nucleotide pool. Finally, while some AID-GFP fused molecules localized to nucleoids, and a significant portion appears to be distributed throughout the bacterial cell, the highest concentration seemed to localize to the cell poles. Chloramphenicol treatment, which detaches the nucleoids from the membrane, caused a further disassociation of AID-GFP from nucleoids suggesting that AID does not intrinsically bind DNA. These results strongly argue against a role for AID in mutagenesis by deamination of cytosine in the nucleotide pool, and suggest that while AID probably acts by deaminating cytosine in the DNA, it requires a protein partner for efficient localization to DNA.
authors.affiliation: Laboratory of Molecular Genetics, Department of Health and Human Services, National Institute of Environmental Health Sciences, National Institutes of Health, 111 TW Alexander Drive, Research Triangle Park, NC 27709, USA. diaz@niehs.nih.gov
authors.forename: Marilyn
authors.initials: M
authors.lastname: Diaz
authors.forename: Madhumita
authors.initials: M
authors.lastname: Ray
authors.forename: Linda J
authors.initials: LJ
authors.lastname: Wheeler
authors.forename: Laurent K
authors.initials: LK
authors.lastname: Verkoczy
authors.forename: Christopher K
authors.initials: CK
authors.lastname: Mathews
mesh_terms: D000071479:APOBEC-1 Deaminase; D000818:Animals; D003564:Cytidine Deaminase / Q000235:genetics / Q000378:metabolism*; D003597:Cytosine Nucleotides / Q000235:genetics / Q000378:metabolism; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D005803:Genes, Immunoglobulin; D017930:Genes, Reporter; D006801:Humans; D016296:Mutagenesis; D051381:Rats
chemical_list: D003597:Cytosine Nucleotides; D000071479:APOBEC-1 Deaminase; C000605089:APOBEC1 protein, human; C000605091:Apobec1 protein, rat; D003564:Cytidine Deaminase