The role of DNA repair proteins in zinc-finger nuclease-induced gene targeting in Drosophila. Ana Bozas

ISBN: 9780549678922

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165 pages


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The role of DNA repair proteins in zinc-finger nuclease-induced gene targeting in Drosophila.  by  Ana Bozas

The role of DNA repair proteins in zinc-finger nuclease-induced gene targeting in Drosophila. by Ana Bozas
| NOOKstudy eTextbook | PDF, EPUB, FB2, DjVu, talking book, mp3, ZIP | 165 pages | ISBN: 9780549678922 | 8.36 Mb

Gene targeting is an important tool in researching gene function, in creating animal models of disease, and ultimately, in gene therapy. The key to our method of gene targeting is using chimeric proteins called zinc fingers nucleases (ZFNs) to createMoreGene targeting is an important tool in researching gene function, in creating animal models of disease, and ultimately, in gene therapy. The key to our method of gene targeting is using chimeric proteins called zinc fingers nucleases (ZFNs) to create double-strand breaks (DSBs).

The ZFNs are comprised of three Cys2His2 zinc fingers as the DNA-binding domain directly linked to the nonspecific nuclease domain of the FokI restriction enzyme.-When used in Drosophila melanogaster, gene targeting with zinc fingers nucleases is a highly efficient process, producing mutants at rates up to 25% of total progeny. A significant proportion of these are directed mutations produced through the homologous recombination (HR) DSB repair pathway.

The remainder of mutations consists of random mutations induced at the targeted locus, and produced through nonhomologous end joining (NHEJ).-In this dissertation I am targeting the rosy locus of D. melanogaster.

I will first show that the majority of the HR repair products depend on a strand invasion process, while only about a 20% remaining fraction of directed mutations are produced through an alternative pathway called single strand annealing.

Secondly, I will show that HR is significantly increased in the absence of the lig4-dependent canonical NHEJ pathway (C-NHEJ), and that the alternative NHEJ repair process can ably take over DSB repair in the absence of all HR and C-NHEJ. In the absence of DNA ligase IV, NHEJ repair products show an increase in nucleolytic processing of DSB ends through the increase in frequency of small deletions at the repair site. Finally, I will show that there is switching between the HR and NHEJ pathways in DSB repair, even at stages as late as ligation, and that this occurs among NHEJ and HR, but not among the sub-branches of these two pathways.-The elucidation of these repair mechanisms will help us in planning future uses of our technology in other model organisms by clarifying the ZFN-specific parameters in the gene targeting process, as well as by giving us insights into the DNA repair mechanisms of Drosophila.



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