Activation-induced deaminase, AID, is catalytically active as a monomer on single-stranded DNA.
Hypermutation and class switch recombination of immunoglobulin genes are antigen-activated mechanisms triggered by AID, a cytidine deaminase. AID deaminates cytidine residues in the DNA of the variable and the switch regions of the immunoglobulin locus. The resulting uracil induces error-prone DNA synthesis in the case of hypermutation or DNA breaks that activate non-homologous recombination in the case of class switch recombination. In vitro studies have demonstrated that AID deaminates single-stranded but not double-stranded substrates unless AID is in a complex with RPA and the substrate is actively undergoing transcription. However, it is not clear whether AID deaminates its substrates primarily as a monomer or as a higher order oligomer. To examine the oligomerization state of AID alone and in the presence of single-stranded DNA substrates of various structures, including loops embedded in double-stranded DNA, we used atomic force microscopy (AFM) to visualize AID protein alone or in complex with DNA. Surprisingly, AFM results indicate that most AID molecules exist as a monomer and that it binds single-stranded DNA substrates as a monomer at concentrations where efficient deamination of single-stranded DNA substrates occur. The rate of deamination, under conditions of excess and limiting protein, also imply that AID can deaminate single-stranded substrates as a monomer. These results imply that non-phosphorylated AID is catalytically active as a monomer on single-stranded DNA in vitro, including single-stranded DNA found in loops similar to those transiently formed in the immunoglobulin switch regions during transcription.
Brar
Sukhdev S
SS
Laboratory of Molecular Genetics, D3-01, National Institute of Environmental Health Sciences/National Institutes of Health, 111 TW Alexander Drive, Research Triangle Park, NC 27709, USA.
Sacho
Elizabeth J
EJ
Tessmer
Ingrid
I
Croteau
Deborah L
DL
Erie
Dorothy A
DA
Diaz
Marilyn
M
D001483:Base Sequence; D002384:Catalysis; D003564:Cytidine Deaminase / Q000378:metabolism*; D017931:DNA Primers; D004261:DNA Replication; D004277:DNA, Single-Stranded / Q000378:metabolism*; D018625:Microscopy, Atomic Force; D013379:Substrate Specificity; D000098830:AICDA (Activation-Induced Cytidine Deaminase)
D017931:DNA Primers; D004277:DNA, Single-Stranded; D000098830:AICDA (Activation-Induced Cytidine Deaminase); D003564:Cytidine Deaminase