Research Article: DNA Damage, Homology-Directed Repair, and DNA Methylation

Date Published: July 6, 2007

Publisher: Public Library of Science

Author(s): Concetta Cuozzo, Antonio Porcellini, Tiziana Angrisano, Annalisa Morano, Bongyong Lee, Alba Di Pardo, Samantha Messina, Rodolfo Iuliano, Alfredo Fusco, Maria R Santillo, Mark T Muller, Lorenzo Chiariotti, Max E Gottesman, Enrico V Avvedimento, Wendy Bickmore

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.

Partial Text: Cellular metabolites and exogenous DNA-damaging agents constantly modify DNA. Such modifications may induce death in unicellular organisms or degenerative changes and aging in multicellular organisms. DNA damage can also activate or amplify biochemical pathways that regulate cell growth and division or coordinate DNA replication with DNA repair. Of the various forms of DNA damage, the most hazardous to the cell are DNA double-strand breaks (DSBs). DSBs are generated when the two complementary strands of the DNA double helix are broken simultaneously at nearby sites. Physically dissociated ends can recombine inappropriately with other genomic sites, leading to chromosomal translocations.

To explore the molecular mechanism(s) linking DNA damage and gene silencing, we induced a DSB at a specific DNA sequence in the genome and monitored recombination and expression of the recombinant gene. We also examined the structure and methylation pattern at the DSB locus following repair. Finally, we asked if inhibiting DNA methylation affected recombination and/or expression of the recombinant product.