Development of a novel system to induce double-strand breaks in mammalian cells.
Doctoral thesis, UCL (University College London).
The aim of this thesis was to develop and set up a novel system in mammalian cells that would help to decipher the complex cellular response to double-strand breaks (DSBs). The system is based on the use of a thermo-sensitive mutant of the rare cutting endonuclease I-SceI. The temperature-controlled cutting activity of such a mutant, combined with transcriptional control of its expression using a tetracycline-dependent promoter, should bring tight control over the induction of a DSB at I-SceI recognition site. By acquiring such control over production of a DSB at an engineered I-SceI recognition site in the mammalian genome, the system should allow the study of the DSB repair kinetics and early events of the DSB repair process. Central to the project was the production of a temperature-dependent mutant of I-SceI. The S. cerevisiae strain KSM1, offered a satisfactory model to screen and select thermo-sensitive phenotypes of the enzyme. The combination of random PCR mutagenesis with the yeast GAP repair mechanism to incorporate the different mutants into a yeast expression plasmid, in a strain carrying an adequate genetic reporter system, all proved to be proficient for selecting temperature dependent I-SceI phenotypes. Mutants with cutting activity at 25°C and 30°C, but with a substantial decrease of activity at 37°C where selected. Most of these selected mutants presented single misense mutations, two mutants presented two and three missense mutations and one beared a nonsense mutation. Four mutants were selected as final candidates and their activities at 30°C and 37°C were tested in a mammalian cell line. Their activities showed remarkably low when compare to wild-type I-SceI activity at 30°C. Although the results obtained in mammalian cells were not in contradiction with the activities observed in S. cerevisiae, they are too low to be applied to the study of DSB repair.
|Title:||Development of a novel system to induce double-strand breaks in mammalian cells|
|Additional information:||Authorisation for digitisation not received|
|UCL classification:||UCL > School of Life and Medical Sciences > Faculty of Population Health Sciences > Institute of Child Health > Department of Infection and Immunity > ICH - Molecular Immunology Unit|
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