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DNA DEAMINATION AND BINDING PROPERTIES OF WILD TYPE AND HIGM2 MUTANTS OF ACTIVATION INDUCED CYTIDINE DEAMINASE
by
Yunxiang Mu
A Dissertation Presented to the
FACULTY OF THE USC GRADUATE SCHOOL
UNIVERSITY OF SOUTHERN CALIFORNIA
In Partial Fulfillment of the
Requirements for the Degree
DOCTOR OF PHILOSOPHY
(MOLECULAR BIOLOGY)
May 2012
Copyright 2012 Yunxiang Mu
Object Description
| Title | DNA deamination and binding properties of wild type and HIGM2 mutants of activation induced cytidine daminase |
| Author | Mu, Yunxiang |
| Author email | yunxianm@usc.edu;biochemistrybiochemistry@gmail.com |
| Degree | Doctor of Philosophy |
| Document type | Dissertation |
| Degree program | Molecular Biology |
| School | College of Letters, Arts And Sciences |
| Date defended/completed | 2012-02-27 |
| Date submitted | 2012-04-05 |
| Date approved | 2012-04-05 |
| Restricted until | 2012-04-05 |
| Date published | 2012-04-05 |
| Advisor (committee chair) | Goodman, Myron F. |
| Advisor (committee member) |
Aparicio, Oscar M. McKenna, Charles |
| Abstract | AID is required for SHM and CSR as an initiator protein. Hyper IgM syndrome 2, a primary immunological deficiency disorder caused by AICDA gene (encoding AID) mutations, is characterized by severe decrease in CSR, which is often accompanied with more or less down-regulated SHM. ❧ Here we purified 23 missense AID HIGM2 mutants, which naturally occur in the human population and a few C terminus truncated mutants, one of which is a C-10 amino acid deletion and is known to only cause a defect in CSR but retains WT level or higher activity in SHM. ❧ All the mutants containing one single point mutation showed no detectable deamination activity on ssDNA substrate except for S43P, L98R, and R174S. Comparison of specific activity indicates that they are about 3, 26, 211 fold less active than WT AID respectively. Although these 3 mutants have reduced deamination specific activity, they have similar mutation spectrum to WT AID. To the contrary ΔC15 and ΔC10 deletion mutants have about 2 fold increase in specific activity, whereas ΔC18 and ΔC17 have reduced specific activity. Sequence alignment indicates that helix 6 is a conserved structural feature of all APOBEC proteins. In agreement with this, a C terminus truncation of 19 or more amino acids disrupted the integrity of helix 6 of AID, resulting in the loss of deamination. ❧ Additionally DNA binding activity of some mutants was tested through rotational anisotropy and their dissociation constants were measured. Compared with WT AID R174S displayed 5 fold less binding affinity and the binding affinity of the ΔC10 is comparable to WT AID. Interestingly when we fit the data points from DNA binding by plotting the change in anisotropy as a function of enzyme concentration, we found that the ssDNA binding of WT AID fit better with cooperativity, which is abrogated in the case of C terminus deletion mutants, for which a rectangular hyperbolic curve is the best fit. ❧ Through the comparison of AID to some artificial mutants of APOBEC3G-CD2 domain of which the structure has been solved, the molecular mechanism of AID mutants and the relationship of structure and functions will be discussed. |
| Keyword | deaminase; apobec; hyper IgM syndrome; class switch recombination; somatic hypermutation |
| 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 |
| Provenance | Electronically uploaded by the author |
| Type | texts |
| Legacy record ID | usctheses-m |
| Rights | Mu, Yunxiang |
| Access conditions | The author retains rights to his/her dissertation, thesis or other graduate work according to U.S. copyright law. Electronic access is being provided by the USC Libraries in agreement with the author, as the original true and official version of the work, but does not grant the reader permission to use the work if the desired use is covered by copyright. It is the author, as rights holder, who must provide use permission if such use is covered by copyright. The original signature page accompanying the original submission of the work to the USC Libraries is retained by the USC Libraries and a copy of it may be obtained by authorized requesters contacting the repository e-mail address given. |
| Repository name | University of Southern California Digital Library |
| Repository address | USC Digital Library, University of Southern California, University Park Campus MC 7002, 106 University Village, Los Angeles, California 90089-7002, USA |
| Repository email | cisadmin@usc.edu |
| Archival file | uscthesesreloadpub_Volume3/etd-MuYunxiang-570.pdf |
Description
| Title | Page 1 |
| Full text | DNA DEAMINATION AND BINDING PROPERTIES OF WILD TYPE AND HIGM2 MUTANTS OF ACTIVATION INDUCED CYTIDINE DEAMINASE by Yunxiang Mu A Dissertation Presented to the FACULTY OF THE USC GRADUATE SCHOOL UNIVERSITY OF SOUTHERN CALIFORNIA In Partial Fulfillment of the Requirements for the Degree DOCTOR OF PHILOSOPHY (MOLECULAR BIOLOGY) May 2012 Copyright 2012 Yunxiang Mu |
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