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http://hdl.handle.net/11375/20279
Title: | Characterizing the interactions of ATP and DNA with the MutL Mismatch Repair protein |
Authors: | Ortiz Castro, Mary |
Advisor: | Guarné, Alba |
Department: | Biochemistry and Biomedical Sciences |
Keywords: | MutL, mismatch repair, ATP, DNA, endonuclease activity |
Publication Date: | 2016 |
Abstract: | The fidelity of DNA replication prevents mutations that may lead to cancer predisposition or neurodegenerative diseases. One mechanism that enhances DNA replication fidelity is DNA mismatch repair, which corrects mismatches and small insertion/deletion loops that have escaped polymerase proofreading. In all eukaryotes and most prokaryotes, MutL (a key mismatch repair protein) has an intrinsic endonuclease activity that nicks the newly synthesized strand and recruits downstream factors to remove and correct errors. It has been proposed that ATP binding promotes a series of conformational changes that induce structural order within MutL and stimulates its endonuclease activity. The C-terminal domain of MutL, which harbors the endonuclease site, does not bind to DNA. This has prevented the molecular characterization of its endonuclease activity. In this thesis, we first show that MutL in B. subtilis exhibits asymmetric conformations similar to yeast and human MutL homologs. We also devise a novel approach to bypass the binding defect of the C-terminal domain by using fusion proteins. We find that these fusions bind to DNA specifically and, in the presence of the processivity clamp, can nick DNA. One of these fusion proteins in particular stimulates the nicking activity much more efficiently than the C-terminal domain alone. This work lays the foundation for the mechanistic characterization of the MutL endonuclease and provides a method to stabilize transient protein-DNA interactions. |
URI: | http://hdl.handle.net/11375/20279 |
Appears in Collections: | Open Access Dissertations and Theses |
Files in This Item:
File | Description | Size | Format | |
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OrtizCastro_Mary_C_finalsubmissionAugust2016_MScThesis-Biochemistry.pdf | 3.35 MB | Adobe PDF | View/Open |
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