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THE KINETIC STUDY OF ENGINEERED MBD DOMAIN INTERACTIONS WITH
METHYLATED DNA:
INSIGHT INTO BINDING OF METHYLATED DNA BY MBD2B
by
Po-Han Chen
A Thesis Presented to the
FACULTY OF THE USC GRADUATE SCHOOL
UNIVERSITY OF SOUTHERN CALIFORNIA
In Partial Fulfillment of the
Requirements for the Degree
MASTER OF SCIENCE
(BIOCHEMISTRY AND MOLECULAR BIOLOGY)
December 2009
Copyright 2009 Po-Han Chen
Object Description
| Title | The kinetic study of engineered MBD domain interactions with methylated DNA: insight into binding of methylated DNA by MBD2b |
| Author | Chen, Po-Han |
| Author email | pohanche@usc.edu; pohanche@gmail.com |
| Degree | Master of Science |
| Document type | Thesis |
| Degree program | Biochemistry & Molecular Biology |
| School | Keck School of Medicine |
| Date defended/completed | 2009-10-28 |
| Date submitted | 2009 |
| Restricted until | Restricted until 24 May. 2011. |
| Date published | 2011-05-24 |
| Advisor (committee chair) | Laird-Offringa, Ite A. |
| Advisor (committee member) |
Haworth, Ian S. Tokes, Zoltan A. |
| Abstract | Mehtyl-CpG binding domain protein 2 and 3 (MBD2 and MBD3) contain methyl-CpG binding domains (MBD) and belong to a family of MBD proteins. Methyl-CpG binding domain protein 3-like-1 (MBD3L1) is a protein with highly homology to MBD2 and MBD3, but it lacks the MBD domain in the N-terminus. A new method, methylated-CpG island recovery assay (MIRA), was developed to purify methylated DNA by using the mixture of MBD2b and MBD3L1. MBD3L1 was found to be able to interact with MBD2b and increase the affinity of MBD2b binding to methylated DNA. However, the mechanism is unknown. Our goal is to explore the mechanism of MBD3L1 involved in MBD2b binding to methylated DNA. We created the engineered 1xMBD and 2xMBDs from MBD2. 2xMBDs showed higher affinities than 1xMBD binding to the single and double fully methylated DNA. The alternative binding model and the rebinding model had been hypothesized to increase the affinities of 2xMBDs, but we observed that the alternative binding model was more preferential for 2xMBDs binding to a fully single methyl-CpG by further studies. In addition, the two linked MBDs were able to contact both methyl-CpGs of cis-methylated DNA, which increased a much more stable binding than trans-methylated DNA. The orientation of 2xMBDs binding suggested the spacing between two MBDs may be an important factor involved in the binding mechanism of MBD2b/MBd3L1 complex. Our findings reveal essential properties of MBDs in binding to methylated DNA. By these results, we can depict a clearer mechanism of how an MBD3b/MBD3L1 complex increases the affinity and binds to methylated DNA. |
| Keyword | MBD protein; methyl-CpG |
| 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-m2759 |
| Rights | Chen, Po-Han |
| 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-Chen-3394 |
| Archival file | uscthesesreloadpub_Volume26/etd-Chen-3394.pdf |
Description
| Title | Page 1 |
| Full text | THE KINETIC STUDY OF ENGINEERED MBD DOMAIN INTERACTIONS WITH METHYLATED DNA: INSIGHT INTO BINDING OF METHYLATED DNA BY MBD2B by Po-Han Chen A Thesis Presented to the FACULTY OF THE USC GRADUATE SCHOOL UNIVERSITY OF SOUTHERN CALIFORNIA In Partial Fulfillment of the Requirements for the Degree MASTER OF SCIENCE (BIOCHEMISTRY AND MOLECULAR BIOLOGY) December 2009 Copyright 2009 Po-Han Chen |
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