Morano, Annalisa (2008) Transcription influences repair-induced DNA methylation. [Tesi di dottorato] (Unpublished)

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Item Type: Tesi di dottorato
Uncontrolled Keywords: DNA damage,gene silencing, homologous-directed repair
Date Deposited: 11 Nov 2009 14:22
Last Modified: 30 Apr 2014 19:35


This work is aimed at the dissection of the molecular mechanism(s) linking DNA damage and gene silencing. To this end, we have developed a genetic system that allows a rapid assessment of homologous-directed repair (HR) of an unique DNA double strand break (DSB). Briefly, we induced a DBS in the genome of HeLa or mouse embryonic stem (ES) cells using the I-SceI restriction endonuclease. Homologous recombination repair by gene conversion, initiated at the site of the double strand break, converts 2 inactivated tandem repeated green fluorescent protein (GFP) genes (DR-GFP) in an intact functional gene. The efficiency of HR, under our conditions, is approximately 2%–4% and can be easily quantified by analyzing GFP+ cells. Half of these recombinants expressed GFP poorly, because GFP gene was silenced. Silencing was rapid and associated with HR and DNA methylation of the recombinant gene, since HeLa DR-GFP treatment with 5-aza-2’-deoxycytidine, a DNA demethylating drug, significantly increased the fraction of GFP expressing cells. Methylation did not alter recombination frequency in both cell types. ES cells deficient in DNA methyl-transferase 1 yielded as many recombinants as wild-type cells, but most of these recombinants expressed GFP robustly. Bisulfite analysis of GFP DNA molecules revealed that approximately half of the HR repaired molecules were de novo methylated, principally at the 3’-end of the DSB in a range of ~300bp. The other half GFP molecules were hypomethylated. Uncleaved and non-homologous repaired molecules did not show changes of the methylation profile. DNA methyl-transferase 1 bound specifically to HR GFP DNA, as revealed by chromatin immunoprecipitation and RNA analysis. HR induced novel methylation profiles on top of the old patterns and contributed to the silencing of GFP expression. Inhibition of transcription by

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