Activation-induced deaminase, AID, is catalytically active as a monomer on single-stranded DNA - PubMed (original) (raw)

Activation-induced deaminase, AID, is catalytically active as a monomer on single-stranded DNA

Sukhdev S Brar et al. DNA Repair (Amst). 2008.

Abstract

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.

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Figures

Figure 1

Figure 1

Sequence of substrates used for deamination assays and predicted structures for the duplexed oligonucleotides.

Figure 2

Figure 2

AID deaminates single-stranded DNA in various configurations. A) Schematic representation of deamination reaction and expected product following incubation of end-labeled oligonucleotide with AID and UNG. B) AID deaminates most oligonucleotides where single-stranded DNA (ssDNA) is exposed but fails to deaminate double-stranded DNA (dsDNA), C) Quantitation of autoradiographs after deamination reactions. The substrate devoid of secondary structure (L-oligo) deaminated best but there was substantial deamination of loop structures over time. All reactions contained 2 nM oligonucleotide and 500 nM AID protein and were repeated at least three times.

Figure 3

Figure 3

Atomic Force Microscopy analysis of the oligomeric state of AID. A) 2 and 3-dimensional view of AID protein alone deposited at a final concentration of 10 nM. B) Representative gel image of the rate of deamination by AID when present at 20 nM concentration acting on 10 nM of L-oligo. The range of deaminated product at 30 minutes from 4 experiments for this ratio of AID protein to oligonucleotide was 32%-49%. C) Volume distributions of AID in the absence of DNA (black bars) and of RNaseA-treated AID in the presence of the L-oligo DNA substrate (grey bars). These results are representative of 5, 10, and 20 nM AID alone (black bars) and of 5, 10, and 20 nM AID in the presence of 10–50 nM of either the L-oligo and the loop substrate oligonucleotide (grey bars). Similar results were obtained when the protein was pre-incubated at 500nM with or without substrate, immediately diluted, and deposited at 10nM concentrations. In all cases, the major peaks in the distributions are consistent with the expected volume of an AID monomer (The predicted volumes (see text) of the monomer: (15 nm3) and dimer (45 nm3) are shown on the x-axis.

Figure 4

Figure 4

RNAse A is required for deamination, but pre-incubation does not alter the rate of deamination. 10 nM of L-oligo was incubated with 500 nM of AID protein without any RNaseA (triangles) or with 400 ng RNaseA added either with the oligo (diamonds) or 30 minutes before the additon of oligo (squares).

Figure 5

Figure 5

Concentration dependence of deamination rates indicates that most of the catalysis on single-stranded DNA oligonucleotide is from the monomer. A) Rate of deamination of 100 nM L-oligo in the presence of 5 nM (diamonds), 20 nM (triangles), 100 nM (circles), and 500 nM (squares) AID. The straight lines are linear fits to data points for times ≤ 20 minutes. The initial rates, which were determined from the linear fits, are 0.15, 0.73, 1.27, and 1.34 min−1 for 5 nM, 20 nM, 100 nM, and 500 nM AID, respectively. B) Rate of deamination of 10 nM L-oligo in the presence of 20 nM (squares) and 500 nM (circles) AID. The curves are first order exponential fits to the data. The error bars represent the standard error from 3 or more experiments.

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