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-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.

Double-strand breaks and the concept of short- and long-term epigenetic memory

“…Though the carcinogens employed were not necessarily direct DSB inducers, this study does provide a strong connection between DNA repair-specific DNA methyltransferase functions, aberrant gene hypermethylation and carcinogenesis. In addition, it reinforces the observations made by Cuozzo et al (2007) and O'Hagan et al (2008) with respect to DNA methyltransferase activity, de novo methylation and possible epigenetic silencing.…”

“…For instance, if a particular modification elicited transient gene silencing during repair, its persistence could be anticipated to keep that gene silenced long-term if not indefinitely, depending on the stability of the modification. This point has already been alluded to with respect to DSB-induced DNA hypermethylation in experimental models (Cuozzo et al 2007;O'Hagan et al 2008). Once establishment of aberrant de novo hypermethylation is achieved, this pattern could conceivably be maintained throughout multiple rounds of replication by the maintenance methyltransferase activity of Dnmt1, and possibly even expanded to further stabilize the repressive mark (Fig.…”

“…In this circumstance, impaired γH2AX induction was not due to failure of DSB induction on account of the absence of DNA methyltransferase activity. Cuozzo et al (2007) demonstrated in both HeLa and murine embryonic stem cells that the site-specific induction of a DSB resulted in repair via homologous recombination, where repaired DNA was marked by de novo methylation, thereby silencing the recombined gene. Dnmt1 was observed to be bound to these recombined chromatin regions.…”

Dynamics of DNA damage response proteins at DNA breaks: a focus on protein modifications

“…DSBs can also form upon replication of DNA molecules containing other DNA lesions, such as DNA SSBs, and are produced by specific nucleases during V(D)J and class switch recombination in vertebrate lymphocytes, meiotic recombination in germ cells, mating type switching in yeast, apoptotic cell death, and retroviral integration (Friedberg et al 2006;Jackson and Bartek 2009). Last, but not least, naturally occurring DSBs at chromosome ends are associated with human cell aging, as they are exposed when telomeres become critically short during replicative senescence (d'Adda di Fagagna et al 2003).…”

“…Other ways of triggering DNA DSB signaling in mammalian cells without the need for any exogenous DNA-damaging agent are telomere shortening in senescent cells and artificial telomere uncapping by inducible loss of function of a component of the Shelterin complex that protects chromosome ends, both of which trigger the formation of DDR foci at telomeres (d'Adda di Fagagna et al 2003;Takai et al 2003). Similar uncapping strategies have been used in yeast (Wellinger 2010).…”