GADD45α inhibition of DNMT1 dependent DNA methylation during homology directed DNA repair

Nucleic Acids Res. 2012 Mar;40(6):2481-93. doi: 10.1093/nar/gkr1115. Epub 2011 Dec 1.

Abstract

In this work, we examine regulation of DNA methyltransferase 1 (DNMT1) by the DNA damage inducible protein, GADD45α. We used a system to induce homologous recombination (HR) at a unique double-strand DNA break in a GFP reporter in mammalian cells. After HR, the repaired DNA is hypermethylated in recombinant clones showing low GFP expression (HR-L expressor class), while in high expressor recombinants (HR-H clones) previous methylation patterns are erased. GADD45α, which is transiently induced by double-strand breaks, binds to chromatin undergoing HR repair. Ectopic overexpression of GADD45α during repair increases the HR-H fraction of cells (hypomethylated repaired DNA), without altering the recombination frequency. Conversely, silencing of GADD45α increases methylation of the recombined segment and amplifies the HR-L expressor (hypermethylated) population. GADD45α specifically interacts with the catalytic site of DNMT1 and inhibits methylation activity in vitro. We propose that double-strand DNA damage and the resulting HR process involves precise, strand selected DNA methylation by DNMT1 that is regulated by GADD45α. Since GADD45α binds with high avidity to hemimethylated DNA intermediates, it may also provide a barrier to spreading of methylation during or after HR repair.

Publication types

  • Research Support, N.I.H., Extramural

MeSH terms

  • Alanine / genetics
  • Amino Acid Substitution
  • Cell Cycle Proteins / metabolism*
  • Chromatin / metabolism
  • DNA (Cytosine-5-)-Methyltransferase 1
  • DNA (Cytosine-5-)-Methyltransferases / antagonists & inhibitors
  • DNA (Cytosine-5-)-Methyltransferases / metabolism*
  • DNA Methylation*
  • Dimerization
  • Genes, Reporter
  • Green Fluorescent Proteins / analysis
  • Green Fluorescent Proteins / genetics
  • HEK293 Cells
  • HeLa Cells
  • Humans
  • Nuclear Proteins / metabolism*
  • Recombinational DNA Repair*

Substances

  • Cell Cycle Proteins
  • Chromatin
  • GADD45A protein, human
  • Nuclear Proteins
  • Green Fluorescent Proteins
  • DNA (Cytosine-5-)-Methyltransferase 1
  • DNA (Cytosine-5-)-Methyltransferases
  • DNMT1 protein, human
  • Alanine