DNA Damage, Homology-Directed Repair, and DNA Methylation

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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-2′-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. Genomic DNA can be modified by cytosine methylation. This epigenetic modification is layered on the primary genetic information and can silence the affected gene. Epigenetic modification has been implicated in cancer and aging. To date, the primary cause and the mechanism leading to DNA methylation are not known. By using a sophisticated genetic system, we have induced a single break in the double helix of the genomes of mouse or human cells. This rupture was repaired by a very precise mechanism: the damaged chromosome pairs and retrieves genetic information from an undamaged and homologous DNA partner. This homology-directed repair was marked in half of the repaired molecules by de novo methylation of cytosines flanking the cut. As a direct consequence, the gene in these repaired molecules was silenced. In the remaining molecules, the recombinant DNA was undermethylated and expressed the reconstituted gene. Since homology-directed repair may duplicate or delete genetic information, epigenetic modification of repaired DNA represents a powerful evolutionary force. If the expression of the repaired gene is harmful, only cells inheriting the silenced copy will survive. Conversely, if the function of the repaired gene is beneficial, cells inheriting the under-methylated copy will have a selective advantage.