1. AID Phosphorylation By 杨潇 李毅捷 2008.7.6

2. Background Introduction  B cells undergo two types of genomic alterations to increase antibody diversity: somatic hypermutation (SHM) class switch recombination (CSR)  Activation-induced cytidine deaminase(AID) initiates SHM and CSR

3. Background Introduction A Brief Mechanism  Deamination by AID  Deaminated DNA is subsequently replicated or repaired by different cellular repair mechanisms (BER, MMR, DSBs etc)

4. Background Introduction Substrate of AID  Linear ssDNA, but not linear dsDNA, serves as an effective AID substrate in vitro. (principal substrate)  How can AID gain efficient access to dsDNA in vivo? Transcription is required; R-loop mechanism RPA/transcription dependent mechanism (RGYW).

5. AID Phosphorylation RPA/transcription dependent mechanism  Native AID isolated from stimulated primary B- cell nuclei is phosphorylated on Ser38 and Tyr184 residues.  Protein kinase A (PKA) was found to interact with AID and appears to be a primary kinase responsible for phosphorylation at Ser38 and possibly at Thr27 residues in vivo and in vitro.

6. AID Phosphorylation RPA/transcription dependent mechanism  phosphorylated AID from activated B cells can interact with RPA, a single strand DNA binding protein.  RPA can target AID to transcribed switch regions. Then the complex can deaminate the non-template strand of RGYW-containing substrates.  The higher density of RGYW sequences in transcribed Ig loci (compared to random regions of the genome) might help facilitate access of AID to these sequences

7. AID Phosphorylation Experiment:  Inhibition of PKA in B cells decrease CSR activity;  Conditional deletion of the regulatory subunit of PKA increase CSR activity;  Mutation of the AID Serine38 to Alanine(the S38A mutation) had no effect on overall AID enzymatic activity on ssDNA in vitro,  But such mutation markedly reduces RPA-dependent dsDNA deamination activity and severely impairs the ability of AID to effect CSR in vivo.

8. AID Phosphorylation What do these indicate? PKA(and phosphorylation) is needed for AID activity in vivo. The mutaton on Ser38 did not adversely affect the catalytic site; The residual CSR activity reflects the R-Loop mechanism (phosphorylation-independent); But still phosphorylation-dependent mechanism is more dominant; AID phosphorylation probably has other functions.

9. AID Phosphorylation Other Implications of AID Phosphorylation  Phosphorylated AID appears to be preferentially present in the nucleus of B cells as opposed to the cytoplasm.  AID exists in an inactive state in cytoplasm, until activation via the B-cell receptor leads to PKA- dependent AID activation. AID is phosphorylated, and then transported into the nucleus, where it binds to a co-factor RPA to effect specific transcriptional events.

10. Function of AID

11. Thank You For Attention By 杨潇 李毅捷 2008.7.6