| Autor: |
Ludivine Litzler, Stephen Methot, Anne-Marie Patenaude, Astrid Zahn, Javier Di Noia |
| Jazyk: |
angličtina |
| Rok vydání: |
2016 |
| Předmět: |
|
| Zdroj: |
Bio-Protocol, Vol 6, Iss 3 (2016) |
| Druh dokumentu: |
article |
| ISSN: |
2331-8325 |
| DOI: |
10.21769/BioProtoc.1724 |
| Popis: |
The enzyme Activation induced deaminase (AID) underpins antibody affinity maturation and isotype switching through its mutagenic activity of deaminating deoxycytidine to deoxyuridine in DNA. Subsequent processing of the deoxyuridine initiates the processes of somatic hypermutation (SHM) and class switch recombination (CSR) in B cells. Structure-function analysis of AID requires sensitive and biologically relevant methods to measure its various activities. Here we describe simple but effective methods to measure 1) the ability of AID to mutate the Escherichia coli genome, which provides an indication of its catalytic activity; 2) the capacity of AID to perform SHM by complementing a derivative of the DT40 chicken B cell line; 3) the ability of AID to perform CSR by complementing AID-deficient primary mouse B cells. The combination of the three methods, accompanied by the necessary analysis of AID subcellular localization and protein expression levels and stability, as controls, allows detailed structure-function interrogation of AID. |
| Databáze: |
Directory of Open Access Journals |
| Externí odkaz: |
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