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.
|Advisor:||Haber, James E.|
|Commitee:||Lovett, Susan T., Marr, Michael T., Symington, Lorraine S.|
|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|
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