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BIOCHEMISTRY AND RECONSTITUTION OF V(D)J RECOMBINATION
IN A PURIFIED SYSTEM
by
Haihui Lu
________________________________________________________________
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
(BIOCHEMISTRY AND MOLECULAR BIOLOGY)
May 2008
Copyright 2008 Haihui Lu
Object Description
| Title | Biochemistry and reconstitution of V(D)J recombination in a purified system |
| Author | Lu, Haihui |
| Author email | haihuilu@usc.edu |
| Degree | Doctor of Philosophy |
| Document type | Dissertation |
| Degree program | Biochemistry & Molecular Biology |
| School | Keck School of Medicine |
| Date defended/completed | 2008-03-12 |
| Date submitted | 2008 |
| Restricted until | Unrestricted |
| Date published | 2008-04-01 |
| Advisor (committee chair) | Lieber, Michael R. |
| Advisor (committee member) |
Hsieh, Chih-Lin Langen, Ralf Zandi, Ebrahim |
| Abstract | Evolved from invertebrate transposons, V(D)J recombination is the vertebrate gene rearrangement process for assembling the antigen receptor genes, which encode immunoglobulins and T cell receptors. This process is essential for adapted immunity, and it is one of the most complex DNA transactions in biology. The recombination events are initiated by cleavage at gene segments by the RAG1:RAG2 complex, which results in hairpin formation at the coding ends. The hairpins are opened by the Artemis:DNA-PKcs complex, and then joined via the NHEJ process. Here we present the biochemical studies of several proteins involved in NHEJ and V(D)J recombination, and a biochemically defined system for the reconstitution of V(D)J recombination in vitro.; We found that the kinase activity of the Artemis:DNA-PKcs complex can be activated by hairpin DNA ends in cis, which then allows the hairpins to be nicked in an asymmetrical manner. The nicking of the hairpin coding ends from all of the 39 functional human VH elements were examined, and our data revealed some sequence-dependent variation in the nicking efficiency and position by Artemis:DNA-PKcs.; An XRCC4-like factor, also called Cernunnos, was recently identified as another important factor of NHEJ as well as V(D)J recombination. Here, we find that purified XLF directly interacts with purified XRCC4:DNA ligase IV complex and stimulates the ligase complex in a direct assay for ligation activity. XLF has DNA binding activity, but this binding is dependent on DNA length in a manner most consistent with orientation of the C-terminal alpha helixes parallel to the DNA helix.; Based on these insights, we have reconstituted many aspects of the antigen receptor diversification of V(D)J recombination using 13 highly purified proteins, thereby permitting variable domain exon assembly using this fully defined system. The features of the recombination sites created by this system include all of the features observed in vivo (nucleolytic resection, P nucleotides, and N nucleotide addition), indictating that most, if not all, of the end modification enzymes have been identified. This in vitro reconstitution system provides a powerful tool to further study the detailed mechanism of V(D)J recombination. |
| Keyword | VDJ recombination; reconstitution; RAG; Artemis; DNA-PKcs; NHEJ |
| 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-m1075 |
| Contributing entity | University of Southern California |
| Rights | Lu, Haihui |
| Repository name | Libraries, University of Southern California |
| Repository address | Los Angeles, California |
| Repository email | cisadmin@dots.usc.edu |
| Filename | etd-Lu-20080401 |
| Archival file | uscthesesreloadpub_Volume14/etd-Lu-20080401.pdf |
Description
| Title | Page 1 |
| Contributing entity | University of Southern California |
| Repository email | cisadmin@dots.usc.edu |
| Full text | BIOCHEMISTRY AND RECONSTITUTION OF V(D)J RECOMBINATION IN A PURIFIED SYSTEM by Haihui Lu ________________________________________________________________ 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 (BIOCHEMISTRY AND MOLECULAR BIOLOGY) May 2008 Copyright 2008 Haihui Lu |
