Identification of new functions for BRCT domains
Thesis (Ph. D.)--Massachusetts Institute of Technology, Dept. of Biology, 2008.
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| Format: | Thesis |
| Language: | eng |
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Massachusetts Institute of Technology
2009
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| Online Access: | http://hdl.handle.net/1721.1/45806 |
| _version_ | 1811083487570034688 |
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| author | Mohammad, Duaa H |
| author2 | Michael B. Yaffe. |
| author_facet | Michael B. Yaffe. Mohammad, Duaa H |
| author_sort | Mohammad, Duaa H |
| collection | MIT |
| description | Thesis (Ph. D.)--Massachusetts Institute of Technology, Dept. of Biology, 2008. |
| first_indexed | 2024-09-23T12:33:51Z |
| format | Thesis |
| id | mit-1721.1/45806 |
| institution | Massachusetts Institute of Technology |
| language | eng |
| last_indexed | 2024-09-23T12:33:51Z |
| publishDate | 2009 |
| publisher | Massachusetts Institute of Technology |
| record_format | dspace |
| spelling | mit-1721.1/458062019-04-12T12:07:48Z Identification of new functions for BRCT domains Mohammad, Duaa H Michael B. Yaffe. Massachusetts Institute of Technology. Dept. of Biology. Massachusetts Institute of Technology. Dept. of Biology. Biology. Thesis (Ph. D.)--Massachusetts Institute of Technology, Dept. of Biology, 2008. "September 2008." Leaf 125 blank. Includes bibliographical references (leaves 119-124). Our lab identified the tandem BRCT domains of PTIP function as a DNA damage responsive phospho binding domain that recognizes proteins phosphorylated by ATM and ATR after DNA damage. The PTIP tandem BRCT domains are responsible for phosphorylation-dependent localization of PTIP into 53BP1- and phospho-H2AX (y-H2AX)-containing nuclear foci, a marker of DNA damage. Here we report that the BRCT domains of PTIP show strong binding to a phosphopeptide corresponding to the C-terminus of y-H2AX. In addition, the BRCT domains of MDC1, MCPH1 and BRCA1 bind to this y-H2AX-like peptide. We are interested in the role of PTIP in the DNA damage response, however, after encountering significant hurdles in generating PTIP knockdown cells, we decided to characterize the budding yeast PTIP homolog, Esc4. Esc4 is a protein that like PTIP contains six BRCT domains. Esc4 was originally identified as an enhancer of silencing of chromatin and a regulator of Tyl transposition. We have found that esc4A arrest in metaphase after DNA damage after low dose MMS treatment. We also found that deletion of a subset Esc4interacting proteins also results in a persistent metaphase arrest. Deletion of Esc4 results in sensitivity to a variety of damaging agents. We have also shown that esc4A cells enter S-phase precociously relative to wild-type cells after synchronization with either alpha-factor or elutriation. In addition, using an adenine reversion assay, we have shown that esc4A are more mutagenic. We have demonstrated that endogenously tagged Esc4p interacts with endogenous PCNA specifically after MMS treatment, but not in undamaged or HU treated cells. We have demonstrated that esc4A have altered PCNA mobility after UV irradiation but not after MMS treatment Concurrently, we have been trying to determine the binding activity of the Esc4p BRCT domains. We have been unable to demonstrate that Esc4p binds to phosphopeptides. However, recent work from the Brown lab has shown that Esc4p fails to be recruited to chromatin after DNA damage in the absence of a histone acetyl-transferase Rttl09. We have demonstrated, using an in vitro binding assay, that the BRCT domains of Esc4p bind to peptides containing acetylated lysines. by Duaa H. Mohammad. Ph.D. 2009-06-30T16:20:24Z 2009-06-30T16:20:24Z 2008 Thesis http://hdl.handle.net/1721.1/45806 319169346 eng M.I.T. theses are protected by copyright. They may be viewed from this source for any purpose, but reproduction or distribution in any format is prohibited without written permission. See provided URL for inquiries about permission. http://dspace.mit.edu/handle/1721.1/7582 145 leaves application/pdf Massachusetts Institute of Technology |
| spellingShingle | Biology. Mohammad, Duaa H Identification of new functions for BRCT domains |
| title | Identification of new functions for BRCT domains |
| title_full | Identification of new functions for BRCT domains |
| title_fullStr | Identification of new functions for BRCT domains |
| title_full_unstemmed | Identification of new functions for BRCT domains |
| title_short | Identification of new functions for BRCT domains |
| title_sort | identification of new functions for brct domains |
| topic | Biology. |
| url | http://hdl.handle.net/1721.1/45806 |
| work_keys_str_mv | AT mohammadduaah identificationofnewfunctionsforbrctdomains |