Morano, Annalisa
(2008)
Transcription influences repair-induced DNA methylation.
[Tesi di dottorato]
(Unpublished)
Item Type: |
Tesi di dottorato
|
Lingua: |
English |
Title: |
Transcription influences repair-induced DNA methylation |
Creators: |
Creators | Email |
---|
Morano, Annalisa | annalisamorano@libero.it |
|
Date: |
1 December 2008 |
Number of Pages: |
80 |
Institution: |
Università degli Studi di Napoli Federico II |
Department: |
Biologia e patologia cellullare e molecolare "L. Califano" |
Scuola di dottorato: |
Medicina molecolare |
Dottorato: |
Patologia e fisiopatologia molecolare |
Ciclo di dottorato: |
21 |
Coordinatore del Corso di dottorato: |
nome | email |
---|
Avvedimento, Vittorio Enrico | avvedim@unina.it |
|
Tutor: |
nome | email |
---|
Avvedimento, Vittorio Enrico | avvedim@unina.it | Gottesman, Maxwell | meg8@columbia.edu |
|
Date: |
1 December 2008 |
Number of Pages: |
80 |
Uncontrolled Keywords: |
DNA damage,gene silencing, homologous-directed repair |
Settori scientifico-disciplinari del MIUR: |
Area 06 - Scienze mediche > MED/03 - Genetica medica Area 06 - Scienze mediche > MED/04 - Patologia generale |
[error in script]
[error in script]
Date Deposited: |
11 Nov 2009 14:22 |
Last Modified: |
30 Apr 2014 19:35 |
URI: |
http://www.fedoa.unina.it/id/eprint/3231 |
DOI: |
10.6092/UNINA/FEDOA/3231 |

Abstract
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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