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A ID P hosphorylation

A ID P hosphorylation. By 杨潇 李毅捷 2008.7.6. 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.

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A ID P hosphorylation

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  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 AIDPhosphorylation • 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

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