We found that AID is catalytically active as a monomer on single-stranded DNA in vitro
, even when the single-stranded DNA region is embedded in double-stranded DNA in the form of loops. We did not observe a significant population of volumes consistent with AID dimers by AFM for any of the protein/oligo concentrations, including concentrations where well over 50% of the oligo is deaminated (, data not shown). Pre-incubation of AID at concentrations up to 500nM also failed to alter the percentage of volumes corresponding to the dimer. In addition, the dependence of the deamination rates on AID concentration is consistent with AID functioning as a monomer for both the experiments in which the AID concentration is lower than the DNA concentration () and for those in which the AID monomer concentration is higher than the DNA concentration (). While these results demonstrate that AID is active as a monomer on single-stranded DNA in vitro
, they do not preclude the possibility that AID may also function as a dimer in vivo
(or in vitro
), where it may be post-translationally modified via phosphorylation [45
] (see below).
Comparison of the rates of deamination on single-stranded DNA loops within double-stranded DNA with deamination rates on substrates devoid of any secondary structure such as the L-oligo indicates that even short loops of single-stranded DNA can be deaminated by AID relatively well, albeit with less efficiency than DNA sequences in which the cytidine is expected to be completely exposed, such as the L-oligo ( and ). These results imply that even at low concentrations, as found in the nucleus of cells expressing AID, monomeric AID could catalyze the deamination of cytidine to uracil as long as it can find a suitable substrate in the form of exposed single-stranded DNA patches or loops. In support of this idea, recent data suggests that AID can bind and deaminate single-stranded DNA in a variety of configurations such as displaced single-stranded DNA loops or patches [64
] and even in negatively supercoiled double-stranded DNA [68
] formed during transcription. These results might explain why AID can access and deaminate DNA in non-natural substrates such as in DNA of E. coli
cells, non-lymphoid cells, and non-Ig genes that are actively transcribed [14
], and wherein B-cell specific post-translational modifications of AID do not occur. If for example, dimerization is optimally promoted when AID is specifically phosphorylated in activated B cells, these results imply that non-phosphorylated monomeric AID in other cells could potentiate inappropriate deamination of dC in non-immunoglubulin gene targets. It will be interesting to determine whether monomeric AID contributes to non-targeted AID-mediated deamination in B cell lymphomas or when expressed in non-B cells. If so, the oligomeric status of AID may contribute yet another layer of regulation similar to transcriptional regulation and intracellular localization that keep cells expressing AID from experiencing its mutagenic activity in non-intended targets [72
]. In addition it is likely that, in B cells, targeting co-factors sharpen AID’s substrate specificity uniquely to the Ig V and CSR regions [75
], although these putative co-factors have yet to be identified.
Our results do not imply that the dimer does not form or is not functional. In the AFM images, a few percent of the molecules have volumes that correspond to the dimer and other higher order oligomeric states, although increasing the concentration from 5 nM to 20 nM or following incubation at 500nM, does not result in any detectable changes in the population of molecules with higher volumes. However, It is clear from our studies that in vitro
, i) the monomer is the predominant species in the absence of substrate (at the concentrations examined), ii) the monomer is catalytically active on single-stranded DNA, and iii) binding to single-stranded DNA substrate does not induce any significant dimerization. A recent study that examined the binding of GST-tagged and His-tagged AID supports our conclusion that AID can function as a monomer [64
]. Specifically, in band-shift assays of AID with a DNA substrate containing a single-stranded bubble, the GST-tagged-AID, which is forced to be a dimer by GST dimerization [76
], causes dramatically greater shifts of the DNA than does the His-tagged-AID, consistent with His-tagged-AID binding to DNA as a monomer.
A recent report by Wang and colleagues, suggests that AID dimerization is required for optimal class switch recombination in vivo
; however, these studies examined co-immunoprecipitation of AID with two different tags from cell extracts, and therefore, the apparent dimerization in these studies could be dimerization mediated by other proteins and not a direct interaction of two AID molecules [77
]. In addition, whether or not this “dimerization” is also required for immunoglobulin hypermutation where deamination of dC’s throughout the length of the V region is evident was not tested. Several other reasons may account for the differences between our results and those reported in the previous study [77
]. Namely, we have found that in its most proximal activity, as a deaminase, monomeric AID can deaminate dC’s in vitro
, whereas class switch recombination in vivo
, may require a higher order oligomeric state of AID to help align specific dC’s in switch regions or for other functionalities. Also, it is possible that AID acts both as a monomer and as a dimer in vivo
, perhaps differentially for hypermutation and class switch recombination; a hypermutation phenotype was not reported in that study for the dimer mutants. While both of these processes require AID, they also likely require different co-factors, occur independently of each other, and target different regions of the heavy chain Ig locus. Finally, it is conceivable that phosphorylation or other post-translational modifications of AID in activated B cells may promote dimerization, while the non-modified form of AID acts as a monomer, with less efficiency. In fact, ectopically-expressed AID is not phosphorylated and yet it can deaminate dC in non-lymphoid cells, but at a lower frequency (14, 44, 69–71).
The observation that AID exhibits catalytic activity as a monomer in vitro
is in contrast to other deaminases studied to date, including the RNA editing enzymes such as Apobec-1, and the adenosine deaminases [78
]. Interestingly, these deaminases require dimerization not only for their enzymatic activity but also for specificity of the substrate. For example, homodimers comprising a wild type monomer and a mutant monomer of ADAR1 or ADRAR2 lose only half the ability to deaminate a non-specific dsRNA substrate, in a sequence-independent manner but lose nearly 70% site-selective activity on natural substrates, such as the RNA encoding serotonin 2C subtype receptor [81
]. Consequently, it is possible that while monomeric AID is catalytically active, a homodimer may confer some kind of substrate specificity wherein interaction between the monomers helps align a specific cytosine residue for deamination. This idea is interesting because deamination of cytosine in an RNA target would require exquisite targeting not only to a particular RNA species, but a particular cytosine in its sequence. Such a level of specificity may require dimerization as seen with RNA editing enzymes such as Apobec-1 [78
]. However, an RNA target for AID has not been identified although indirect evidence supports the possibility [reviewed in 38
]. Alternatively, an AID homodimer, while not required for deamination, may help the simultaneous deamination of cytosine on both DNA strands, as previously suggested [49
], an idea supported by our findings predicting that each monomer of AID in a putative dimer can catalyze deamination of single-stranded DNA independently. Additional support for this notion comes from theoretical analysis based on a comparison of yeast RNA-editing deaminase to AID suggesting that for the homodimer, both monomers are able to catalyze deamination of two cytosines within the same nucleic acid strand or in opposite strands [51
]. In addition, a recent structure of the AID homolog APOBEC2 shows that while APOBEC2 forms a tetramer, which is a dimer of dimers, the putative active site is not near either the dimer or tetramer interface and each of the monomers appears to have a fully intact active site [82
]. Finally, it is important to note that although it is likely that AID can form a dimer, there is no direct evidence for dimerization in vitro
or in vivo
[other than the small population of dimers observed in this study ()], only inference from homology models and co-immunoprecipitation of cell extracts [51
]. It will be interesting to determine whether AID catalyzes deamination as a monomer and/or dimer in vivo
, and whether the oligomeric state of AID contributes a previously unappreciated layer of functional plasticity by modifying substrate specificity differentially for SHM or CSR, or for DNA vs RNA substrates depending on whether a specific cytosine needs to be deaminated (as seen in RNA deamination) versus several cytosines in a DNA substrate with some preference (i.e. hotspots) but without requiring deamination of a specific residue.