DNA Damage, Homology-Directed Repair, and DNA Methylation.

@article{citeulike:1459344, 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.}, author = {Cuozzo, Concetta and Porcellini, Antonio and Angrisano, Tiziana and Morano, Annalisa and Lee, Bongyong and Pardo, Alba Di D. and Messina, Samantha and Iuliano, Rodolfo and Fusco, Alfredo and Santillo, Maria R R. and Muller, Mark T T. and Chiariotti, Lorenzo and Gottesman, Max E E. and Avvedimento, Enrico V V. }, citeulike-article-id = {1459344}, doi = {http://dx.doi.org/10.1371/journal.pgen.0030110}, issn = {1553-7404}, journal = {PLoS Genet}, keywords = {methylation}, month = {July}, number = {7}, posted-at = {2008-05-01 16:47:28}, priority = {2}, title = {DNA Damage, Homology-Directed Repair, and DNA Methylation.}, url = {http://dx.doi.org/10.1371/journal.pgen.0030110}, volume = {3}, year = {2007} }

TY - JOUR ID - citeulike:1459344 L3 - citeulike-article-id:1459344 TI - DNA Damage, Homology-Directed Repair, and DNA Methylation. JF - PLoS Genet VL - 3 IS - 7 SN - 1553-7404 N2 - 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. KW - methylation AU - Cuozzo, Concetta AU - Porcellini, Antonio AU - Angrisano, Tiziana AU - Morano, Annalisa AU - Lee, Bongyong AU - Pardo, Alba Di AU - Messina, Samantha AU - Iuliano, Rodolfo AU - Fusco, Alfredo AU - Santillo, Maria R AU - Muller, Mark T AU - Chiariotti, Lorenzo AU - Gottesman, Max E AU - Avvedimento, Enrico V PY - 2007/07/06/ UR - http://dx.doi.org/10.1371/journal.pgen.0030110 ER -