Show simple item record

dc.contributor.advisorKendall L. Knight, Ph.D.
dc.contributor.authorForget, Anthony L.
dc.date2022-08-11T08:08:44.000
dc.date.accessioned2022-08-23T16:06:18Z
dc.date.available2022-08-23T16:06:18Z
dc.date.issued2004-04-17
dc.date.submitted2006-07-19
dc.identifier.doi10.13028/edwz-yd18
dc.identifier.urihttp://hdl.handle.net/20.500.14038/32034
dc.description<p>Some images did not scan well. Please see print version for images.</p>
dc.description.abstractThe error free repair of DNA double strand breaks through the homologous recombinational repair pathway is essential for organisms of all types to sustain life. A detailed structural and mechanistic understanding of this pathway has been the target of intense study since the identification of bacterial recA, the gene whose product is responsible for the catalysis of DNA strand exchange, in 1965. The work presented here began with defining residues that are important for the assembly and stability of the RecA filament, and progressed to the identification of residues critical for the transfer of ATP-mediated allosteric information between subunits in the protein's helical filament structure. My work then evolved to investigate similar mechanistic details concerning the role of ATP in the human RecA homolog, Rad51. Results from non-conservative mutagenesis studies of the N-terminal region of one subunit and the corresponding interacting surface on the neighboring subunit within the RecA protein, led to the identification of residues critical for the formation of the inactive RecA filament but not the active nucleoprotein filament. Through the use of specifically engineered cysteine substitutions we observed an ATP-induced change in the efficiency of cross subunit disulfide bond formation and concluded that the position of residues in this region as defined by the current crystal structure may not accurately reflect the active form of the protein. These ATP induced changes in positioning led to the further investigation of the allosteric mechanism resulting in the identification of residue Phe217 as the key mediator for ATP-induced information transfer from one subunit to the next. In transitioning to investigate homologous mechanisms in the human pathway I designed a system whereby we can now analyze mutant human proteins in human cells. This was accomplished through the use of RNA interference, fluorescent transgenes, confocal microscopy and measurements of DNA repair. In the process of establishing the system, I made the first reported observation of the cellular localization of one of the Rad51 paralogs, Xrcc3, before and after DNA damage. In addition we found that a damage induced reorganization of the protein does not require the presence of Rad51 and the localization to DNA breaks occurs within 10 minutes. In efforts to characterize the role of ATP in human Rad51 mediated homologous repair of double strand breaks we analyzed two mutations in Rad51 specifically affecting ATP hydrolysis, K133A and K133R. Data presented here suggests that, in the case of human cells, ATP hydrolysis and therefore binding, by Rad51 is essential for successful repair of induced damage.
dc.language.isoen_US
dc.rightsCopyright is held by the author, with all rights reserved.
dc.subjectDNA-Binding Proteins
dc.subjectDNA Repair
dc.subjectRec A Recombinases
dc.subjectRecombinant Proteins
dc.subjectAmino Acids, Peptides, and Proteins
dc.subjectGenetic Phenomena
dc.titleHomologous Recombinational DNA Repair: from Prokaryotes to Eukaryotes: a Dissertation
dc.typeDoctoral Dissertation
dc.identifier.legacyfulltexthttps://escholarship.umassmed.edu/cgi/viewcontent.cgi?article=1068&amp;context=gsbs_diss&amp;unstamped=1
dc.identifier.legacycoverpagehttps://escholarship.umassmed.edu/gsbs_diss/68
dc.legacy.embargo2017-04-24T00:00:00-07:00
dc.identifier.contextkey181765
refterms.dateFOA2022-08-26T04:23:25Z
html.description.abstract<p>The error free repair of DNA double strand breaks through the homologous recombinational repair pathway is essential for organisms of all types to sustain life. A detailed structural and mechanistic understanding of this pathway has been the target of intense study since the identification of bacterial <em>recA</em>, the gene whose product is responsible for the catalysis of DNA strand exchange, in 1965. The work presented here began with defining residues that are important for the assembly and stability of the RecA filament, and progressed to the identification of residues critical for the transfer of ATP-mediated allosteric information between subunits in the protein's helical filament structure. My work then evolved to investigate similar mechanistic details concerning the role of ATP in the human RecA homolog, Rad51.</p> <p>Results from non-conservative mutagenesis studies of the N-terminal region of one subunit and the corresponding interacting surface on the neighboring subunit within the RecA protein, led to the identification of residues critical for the formation of the inactive RecA filament but not the active nucleoprotein filament. Through the use of specifically engineered cysteine substitutions we observed an ATP-induced change in the efficiency of cross subunit disulfide bond formation and concluded that the position of residues in this region as defined by the current crystal structure may not accurately reflect the active form of the protein.</p> <p>These ATP induced changes in positioning led to the further investigation of the allosteric mechanism resulting in the identification of residue Phe217 as the key mediator for ATP-induced information transfer from one subunit to the next.</p> <p>In transitioning to investigate homologous mechanisms in the human pathway I designed a system whereby we can now analyze mutant human proteins in human cells. This was accomplished through the use of RNA interference, fluorescent transgenes, confocal microscopy and measurements of DNA repair. In the process of establishing the system, I made the first reported observation of the cellular localization of one of the Rad51 paralogs, Xrcc3, before and after DNA damage. In addition we found that a damage induced reorganization of the protein does not require the presence of Rad51 and the localization to DNA breaks occurs within 10 minutes.</p> <p>In efforts to characterize the role of ATP in human Rad51 mediated homologous repair of double strand breaks we analyzed two mutations in Rad51 specifically affecting ATP hydrolysis, K133A and K133R. Data presented here suggests that, in the case of human cells, ATP hydrolysis and therefore binding, by Rad51 is essential for successful repair of induced damage.</p>
dc.identifier.submissionpathgsbs_diss/68
dc.contributor.departmentBiochemistry and Molecular Pharmacology
dc.description.thesisprogramBiochemistry and Molecular Pharmacology


Files in this item

Thumbnail
Name:
Forget_Anthony.pdf
Size:
6.532Mb
Format:
PDF

This item appears in the following Collection(s)

Show simple item record