DNA damage, homology-directed repair and DNA methylation

Abstract

To explore the link between DNA damage and gene silencing, we induced a DNA double-strand break in the genome of Hela or mouse embryonic stem (ES) cells using I-SceI restriction endonuclease. The I-SceI site lies within one copy of two inactivated tandem repeated green fluorescent protein (GFP) genes (DR-GFP). A total of 2%-4% of the cells generated a functional GFP by homology-directed repair (HR) and gene conversion. However, ;50% of these recombinants expressed GFP poorly. Silencing was rapid and associated with HR and DNA methylation of the recombinant gene, since it was prevented in Hela cells by 5-aza-29-deoxycytidine. ES cells deficient in DNA methyl transferase 1 yielded as many recombinants as wild-type cells, but most of these recombinants expressed GFP robustly. Half of the HR DNA molecules were de novo methylated, principally downstream to the double-strand break, and half were undermethylated relative to the uncut DNA. Methylation of the repaired gene was independent of the methylation status of the converting template. The methylation pattern of recombinant molecules derived from pools of cells carrying DR-GFP at different loci, or from an individual clone carrying DR-GFP at a single locus, was comparable. ClustalW analysis of the sequenced GFP molecules in Hela and ES cells distinguished recombinant and nonrecombinant DNA solely on the basis of their methylation profile and indicated that HR superimposed novel methylation profiles on top of the old patterns. Chromatin immunoprecipitation and RNA analysis revealed that DNA methyl transferase 1 was bound specifically to HR GFP DNA and that methylation of the repaired segment contributed to the silencing of GFP expression. Taken together, our data support a mechanistic link between HR and DNA methylation and suggest that DNA methylation in eukaryotes marks homologous recombined segments.Citation: Cuozzo C, Porcellini A, Angrisano T, Morano A, Lee B, et al. (2007) DNA damage, homology-directed repair, and DNA methylation. PLoS Genet 3(7): e110.

The emerging role of Polycomb repressors in the response to DNA damage

“…One such mechanism is local DSBinduced silencing. The existence of this phenomenon was demonstrated using stably integrated constructs that allow the induction of a defined DNA break in the direct vicinity of a transcriptional reporter gene (Cuozzo et al, 2007;O'Hagan et al, 2008;. The physiological relevance of these findings has recently been supported by ChIP-based studies that mapped the distribution of both cH2AX and Pol II in parallel with transcription occurring around endonuclease-induced breaks (Iacovoni et al, 2010;Pankotai et al, 2012).…”

New therapeutic perspectives in CCDC6 deficient lung cancer cells

“…S3). To validate this finding in a different reporter model, we compared HDR efficiencies in wild type and knock down CCDC6 cells using a well established HeLa GFP reporter system (containing DR GFP, 25,[31][32][33][34] ). In this reporter, I-SceI expression was placed under control of a teton promoter.…”

“…31 At 48 hr post-transfection, HDR efficiency was determined by evaluating the percentage of GFP1 cells by Flow Cytometry. Cells transfected with or without the I-SceI plasmid revealed that the CCDC6 deficient lines yielded significantly lower GFP1 cells compared to H1975 cells (Fig.…”

Platinum-Based Chemotherapy Induces Methylation Changes in Blood DNA Associated with Overall Survival in Patients with Ovarian Cancer

“…Recent evidence has suggested that mismatch repair proteins at sites of DNA damage including double strand breaks can recruit the DNA methylating enzyme DNMT1 and result in aberrant methylation (2)(3)(4)(5). DNA mismatch repair proteins recognize and bind to sites of platinum-induced DNA damage.…”