Dissertation/Thesis Abstract

Gene targeting occurs through multiple recombination pathways and is differentially sensitive to chromatin state
by Dykstra, Sarah G., Ph.D., Brandeis University, 2015, 329; 3687263
Abstract (Summary)

Gene targeting is a process in which a double stranded linear DNA fragment is introduced into a genome. This homologous recombination event is dependent on members of the RAD52 epistasis family; however the role that these genes play in targeted gene replacement remains an area of investigation. In this thesis, I describe the development and characterization of a haploid S. cerevisiae system to study the pathways involved in targeted gene replacement. Using this system, I have determined that gene targeting requires POL32 for most events and occurs through alternate RAD51- and RAD59- independent pathways as well as an as yet undetermined RAD51- and RAD59- independent, but RAD52-dependent pathway. Furthermore, I have determined that each of these pathways is mechanistic unique and has different genetic requirements. Finally, I have shown each off these pathways is differentially sensitive to chromatin state as RAD51-independent gene targeting preferentially recombines at sites of heterochromatin, while RAD59 -independent gene targeting occurs more frequently at sites of euchromatin. Taken together, these findings provide insight into the mechanism(s) of gene targeting and may provide a basis for enhancing gene targeting efficiency in other organisms and contexts.

Indexing (document details)
Advisor: Haber, James E.
Commitee: Lovett, Susan T., Marr, Michael T., Symington, Lorraine S.
School: Brandeis University
Department: Molecular and Cell Biology
School Location: United States -- Massachusetts
Source: DAI-B 76/08(E), Dissertation Abstracts International
Subjects: Molecular biology, Genetics
Keywords: Chromatin, Gene targeting, Homologous recombination, Rad51, Rad59, Targeted gene replacement
Publication Number: 3687263
ISBN: 978-1-321-64776-1
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