Impact of Phosphorylation and Phosphorylation-null Mutants on the Activity and Deamination Specificity of Activation-induced Cytidine Deaminase

Abstract

Activation-induced cytidine deaminase (AID) initiates somatic hypermutation and class switch recombination in B cells by deaminating C 3 U on transcribed DNA. Here we analyze the role of phosphorylation and phosphorylation-null mutants on the biochemical behavior of AID, including enzyme specific activity, processivity, deamination spectra, deamination motif specificity, and transcription-dependent deamination in the presence and absence of RPA. We show that a small fraction of recombinant human AID expressed in Sf9 insect cells is phosphorylated at previously identified residues Ser 38 and Thr 27 and also at Ser 41 and Ser 43 . S43P AID has been identified in a patient with hyper-IgM immunodeficiency syndrome. Ser-substituted phosphorylation-null mutants (S38A, S41A, S43A, and S43P) exhibit wild type (WT) activity on single-stranded DNA. Deamination of transcribed double-stranded DNA is similar for WT and mutant AID and occurs with or without RPA. Although WT and AID mutants catalyze processive deamination favoring canonical WRC hot spot motifs (where W represents A/T and R is A/G), their deamination spectra differ significantly. The differences between the WT and AID mutants appear to be caused by the replacement of Ser as opposed to an absence of phosphorylation. The spectral differences reflect a marked change in deamination efficiencies in two motifs, GGC and AGC, which are preferred by mutant AID but disfavored by WT AID. Both motifs occur with exceptionally high frequency in human switch regions, suggesting a possible relationship between AID deamination specificity and a loss of antibody diversification.AID, a B-cell specific protein, is required for tightly regulated mechanisms of Ig antibody diversification, somatic hypermutation, class switch recombination, and gene conversion. SHM 2 is characterized by an exceptionally high mutation rate ϳ10 Ϫ3 to 10 Ϫ4 per base pair per cell division within the V(D)J rearranged Ig genes (1). CSR is a unique region specific event in which the donor switch region upstream of the IgH C exon (coding for an IgM antibody) recombines with one of the switch regions that is 5Ј to each of the downstream C exons, such as C ␥ , C ␣ , or C ⑀ , producing IgG, IgA, or IgE antibody isotypes, respectively (2).Typically, under tight transcriptional control, AID is induced in activated B cells within germinal centers, causing deamination of cytosine residues in the variable and switch regions (Vand S-regions, respectively) of transcribed Ig loci. Deaminated DNA is subsequently replicated or repaired by different cellular repair mechanisms to give rise to diversified isotype-switched and antigen-specific high affinity antibodies (for reviews, see .Biochemical studies have shown that purified native B-cell AID or recombinant AID protein, expressed in Sf9 insect cells or Escherichia coli, deaminates C residues on ssDNA but not dsDNA, single-stranded RNA, or DNA/RNA hybrid molecules (6 -9). AID acts processively on ssDNA and has a distinctive deamination specificity, favoring C targets in...

Stochastic properties of processive cytidine DNA deaminases AID and APOBEC3G

“…We have found that AID expressed in Sf 9 insect cells can be phosphorylated on Ser 38 and Thr27 as well as two additional phosphorylation sites, Ser41 and Ser43 (Pham et al 2008). Analysis of this mass spectrometry data suggests that only one residue can be phosphorylated on AID at a time (Pham et al 2008).…”

“…This suggests that the jumping motion of APOBEC3G contributes to a significant portion of the processivity (Chelico et al 2006(Chelico et al , 2008. Deamination spectra of AID show large gaps between clustered deamination sites suggesting that the enzyme can jump and slide (Pham et al 2003(Pham et al , 2008Bransteitter et al 2004). Sliding seems to permit deamination of closely spaced residues whereas jumping would facilitate reaching multiple regions of an ssDNA substrate (figure 2c).…”

“…Although phosphorylation does not appear to be B-cell specific (McBride et al 2006), AID phosphorylation at Ser38 was suggested to be the modification targeting AID to transcribed dsDNA through interactions with replication protein A (RPA) (Chaudhuri et al 2004;Basu et al 2005). On the other hand, recombinant AID, expressed in E. coli or Sf 9 insect cells, has been shown to deaminate transcribed dsDNA in the absence of RPA (Pham et al 2003(Pham et al , 2008Sohail et al 2003;Bransteitter et al 2004;Shen & Storb 2004;Besmer et al 2006). Since the phosphorylation mutant S38A has differing levels of CSR inhibition in vivo (Basu et al , 2007Pasqualucci et al 2006;Shinkura et al 2007), we have carried out a comparative biochemical analysis using WT AID, S38A and additional phosphorylation defective mutants, to examine the alternative roles of phosphorylation that could account for a loss of AID CSR activity in vivo.…”

“…(c) The stochastic nature of three-dimensional processivity The data generated from these processivity experiments ( figure 2a(i)) can also be used to calculate the probability that if AID deaminates at the 5 0 -end target it will move on to deaminate the 3 0 -end target and vice versa (Chelico et al 2006;Pham et al 2008). This serves as a measure of AID's correlated deamination efficiency.…”

“…AID-catalysed deaminations on transcribed dsDNA must be highly dynamic to follow along with the moving transcription bubble. Nevertheless, AID retains its key biochemical properties on transcribed dsDNA substrates: a preference for 5 0 -WRC hot-spot motifs and diverse clonal mutagenic distribution (Bransteitter et al 2004;Pham et al 2008). AID, purified from mouse B cells, has been shown to be phosphorylated on Ser38, Tyr184 and possibly Thr27 McBride et al 2006;Pasqualucci et al 2006).…”