Paper: DNA Damage, Homology-Directed Repair, and DNA Methylation

DNA Damage, Homology-Directed Repair, and DNA Methylation

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

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.

Source: PLoS Genetics (Open Access)
http://genetics.plosjournals.org/perlserv/?request=get-document&doi=10.1371/journal.pgen.0030110

Posted by
Robert Karl Stonjek


.

Relevant Pages

  • Re: Death by Ignorance
    ... lot to reduce the incidence of disease and increase our lifespan. ... Researchers at Johns Hopkins have found that epigenetic marks on DNA-chemical marks other than the DNA sequence-do indeed change ... "We're beginning to see that epigenetics stands at the center of modern medicine because epigenetic changes, ... "Inappropriate methylation levels can contribute to disease-too much might turn necessary genes off, too little might turn genes on ...
    (soc.retirement)
  • Re: Death by Ignorance
    ... And May Explain Many 'Late-onset' Diseases ... Researchers at Johns Hopkins have found that epigenetic marks on DNA-chemical marks other than the DNA sequence-do indeed change ... "We're beginning to see that epigenetics stands at the center of modern medicine because epigenetic changes, ... "Inappropriate methylation levels can contribute to disease-too much might turn necessary genes off, too little might turn genes on ...
    (soc.retirement)
  • Article: In A Technical Tour De Force, Scientists Take A Global View Of The Epigenome
    ... genome-wide DNA methylation pattern of the plant Arabidopsis thaliana - the ... full of plant genes were known to be regulated by methylation. ... stem cells differs from that in a mature skin or nerve cell. ...
    (sci.bio.evolution)
  • Re: Changes to DNA
    ... Basically diet doesn't change the *DNA*, ... This is called the "methylation patterns" ... Whilst I understand your explanation that diet does not have a *direct* ...
    (talk.origins)
  • Re: Evolution Strawbuster
    ... I seem to recall that I said something like "the DNA which codes for ... ten genes or half a gene or whatever, ... Eubacteria it shows an unresolved 24-way node which is absurd, ... Eukarya, their metabolic genes are more like those of Eubacteria. ...
    (talk.origins)