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HOW PHOSPHORYLATION AFFECTS THE BIOCHEMICAL
REGULATION AND TARGETING OF ACTIVATION INDUCED
CYTIDINE DEAMINASE (AID)
by
Sariah J. Allen
A Dissertation Presented to the
FACULTY OF THE GRADUATE SCHOOL
UNIVERSITY OF SOUTHERN CALIFORNIA
In Partial Fulfillment of the
Requirements for the Degree
DOCTOR OF PHILOSOPHY
(MOLECULAR BIOLOGY)
December 2007
Copyright 2007 Sariah J. Allen
Object Description
| Title | How phosphorylation affects the biochemical regulation and targeting of activation induced cytidine deaminase (AID) |
| Author | Allen, Sariah J. |
| Author email | sallen@usc.edu |
| Degree | Doctor of Philosophy |
| Document type | Dissertation |
| Degree program | Molecular & Computational Biology |
| School | College of Letters, Arts and Sciences |
| Date defended/completed | 2007-11-20 |
| Date submitted | 2007 |
| Restricted until | Unrestricted |
| Date published | 2007-12-05 |
| Advisor (committee chair) | Quick, Michael |
| Advisor (committee member) |
Lemus, Judith Chen, Xiaojiang |
| Abstract | Activation induced cytidine deaminase (AID) is required for somatic hypermutation (SHM) and class-switch recombination (CSR). AID initiates the processes that carry out immunoglobulin diversity by deaminating cytosine residues within variable (V) and switch (S) regions on the Ig locus during active transcription. The resulting G:U mispairs can then be replicated or repaired by cellular repair mechanisms to give rise to isotype-switched and antigen-specific mature antibodies.; In this study I have identified two novel phosphorylation sites, serine 41 and serine 43, and demonstrated their importance in AID activity as well as confirmed the importance of serine 38 phosphorylation. Phosphorylation null mutants generated by replacing serine with alanine are much less active than wild-type AID, as is non-phosphorylated AID purified from E. coli. In contrast, phosphorylation charge mimic mutants generated by replacing serine with aspartic acid, are (3-4) fold more active than wild-type AID. Phosphorylation does not affect the ability of AID to deaminate actively transcribed double-stranded DNA or its ability to preferentially deaminate WRC hot spots. No observed affect on DNA binding strength was detected suggesting that mutant enzymes are folded like wild-type AID.; The highly conserved nature of these phosphorylation sites and the identification of Hyper-IgM Type 2 (HIGM2) patients with mutations at serine 43, taken together with our biochemical data suggest that these phosphorylation sites are required for proper AID function in B cells. I propose a model whereby AID phosphorylation is involved in the regulation of AID activity and efficient targeting during SHM and CSR. Additionally, I propose that phosphorylation serves as a gatekeeper for genomic integrity, protecting the B cell genome against inappropriate deamination by providing a rapid activation and targeting mechanism. |
| Keyword | AID; SHM; CSR; antibody divsersification; activation induced cytidine deaminase |
| Language | English |
| Part of collection | University of Southern California dissertations and theses |
| Publisher (of the original version) | University of Southern California |
| Place of publication (of the original version) | Los Angeles, California |
| Publisher (of the digital version) | University of Southern California. Libraries |
| Type | texts |
| Legacy record ID | usctheses-m966 |
| Contributing entity | University of Southern California |
| Rights | Allen, Sariah J. |
| Repository name | Libraries, University of Southern California |
| Repository address | Los Angeles, California |
| Repository email | http://www.usc.edu/isd/libraries/services/ask_a_librarian/email/ |
| Filename | etd-Allen-20071205 |
| Archival file | uscthesesreloadpub_Volume44/etd-Allen-20071205.pdf |
Description
| Title | Page 1 |
| Contributing entity | University of Southern California |
| Full text | HOW PHOSPHORYLATION AFFECTS THE BIOCHEMICAL REGULATION AND TARGETING OF ACTIVATION INDUCED CYTIDINE DEAMINASE (AID) by Sariah J. Allen A Dissertation Presented to the FACULTY OF THE GRADUATE SCHOOL UNIVERSITY OF SOUTHERN CALIFORNIA In Partial Fulfillment of the Requirements for the Degree DOCTOR OF PHILOSOPHY (MOLECULAR BIOLOGY) December 2007 Copyright 2007 Sariah J. Allen |
