Mutations in human microcephalin (MCPH1) result in a form of autosomal recessive primary microcephaly, a disorder of fetal brain growth characterized by a severely reduced cerebral cortex and head size with mental retardation. Both human and Drosophila MCPH1 contain BRCA1 C-terminal domains (BRCT domains), which are found in many proteins that function in DNA repair and cell-cycle control. Maternal-effect lethal mutations in Drosophila mcph1 result in cell-cycle arrest due to triggering of the centrosome inactivation pathway, a Checkpoint kinase 2 (Chk2)-mediated response following DNA damage or incomplete DNA replication in early Drosophila embryos (Rickmyre et al. 2007). mcph1 embryos exhibit genomic instability as evidenced by frequent chromatin bridging in anaphase. Furthermore, in contrast to studies of human MCPH1, the ATR/Chk1-mediated DNA checkpoint appears to be intact in Drosophila mcph1 mutants. In order to further understand how MCPH1 functions, I used tandem affinity purification (TAP)-mass spectrometry to find interactors. This approach revealed several regulators of chromatin structure, RNAi machinery components, and proteins involved in DNA replication in complex with Drosophila MCPH1.
In collaboration with Dr. Marc Kirschners laboratory, we identified Drosophila MCPH1 in a genome-scale biochemical screen for substrates of the Anaphase-Promoting Complex (APC). We have identified the destruction box of MCPH1 required for APC-mediated degradation in vitro. Our in vivo data indicate that MCPH1 protein levels are elevated in embryos from females carrying a maternal-effect allele of APC2, which encodes the functional ligase of the APC. We have also demonstrated that human MCPH1 is an in vitro substrate of the APC and have shown that its levels oscillate in a cell-cycle dependent manner in cultured human cells with lower protein levels when APC is activated during late mitosis and G1. Finally, injection of 2- or 4-cell staged Xenopus embryos with human or Drosophila mcph1 RNA results in cell-cycle arrest. Injected cells undergo a few rounds of normal cleavage before exhibiting failed cytokinesis while the uninjected cells continue to divide unperturbed.
Identifer | oai:union.ndltd.org:VANDERBILT/oai:VANDERBILTETD:etd-07262010-135944 |
Date | 05 August 2010 |
Creators | Rickmyre, Jamie Lyn |
Contributors | Kathleen Gould, David Cortez, Ethan Lee, Laura Lee |
Publisher | VANDERBILT |
Source Sets | Vanderbilt University Theses |
Language | English |
Detected Language | English |
Type | text |
Format | application/pdf |
Source | http://etd.library.vanderbilt.edu/available/etd-07262010-135944/ |
Rights | unrestricted, I hereby certify that, if appropriate, I have obtained and attached hereto a written permission statement from the owner(s) of each third party copyrighted matter to be included in my thesis, dissertation, or project report, allowing distribution as specified below. I certify that the version I submitted is the same as that approved by my advisory committee. I hereby grant to Vanderbilt University or its agents the non-exclusive license to archive and make accessible, under the conditions specified below, my thesis, dissertation, or project report in whole or in part in all forms of media, now or hereafter known. I retain all other ownership rights to the copyright of the thesis, dissertation or project report. I also retain the right to use in future works (such as articles or books) all or part of this thesis, dissertation, or project report. |
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