Tyrosine phosphorylation enhances RAD52-mediated annealing by modulating its DNA binding

EMBO J. 2011 Jul 29;30(16):3368-82. doi: 10.1038/emboj.2011.238.


RAD52 protein has an important role in homology-directed DNA repair by mediating RAD51 nucleoprotein filament formation on single-stranded DNA (ssDNA) protected by replication protein-A (RPA) and annealing of RPA-coated ssDNA. In human, cellular response to DNA damage includes phosphorylation of RAD52 by c-ABL kinase at tyrosine 104. To address how this phosphorylation modulates RAD52 function, we used an amber suppressor technology to substitute tyrosine 104 with chemically stable phosphotyrosine analogue (p-Carboxymethyl-L-phenylalanine, pCMF). The RAD52(Y104pCMF) retained ssDNA-binding activity characteristic of unmodified RAD52 but showed lower affinity for double-stranded DNA (dsDNA) binding. Single-molecule analyses revealed that RAD52(Y104pCMF) specifically targets and wraps ssDNA. While RAD52(Y104pCMF) is confined to ssDNA region, unmodified RAD52 readily diffuses into dsDNA region. The Y104pCMF substitution also increased the ssDNA annealing rate and allowed overcoming the inhibitory effect of dsDNA. We propose that phosphorylation at Y104 enhances ssDNA annealing activity of RAD52 by attenuating dsDNA binding. Implications of phosphorylation-mediated activation of RAD52 annealing activity are discussed.

Publication types

  • Comparative Study
  • Research Support, N.I.H., Extramural
  • Research Support, Non-U.S. Gov't
  • Research Support, U.S. Gov't, Non-P.H.S.

MeSH terms

  • Amino Acid Sequence
  • Amino Acid Substitution
  • Amino Acids / metabolism
  • Binding, Competitive
  • DNA / metabolism
  • DNA / pharmacology
  • DNA Breaks, Double-Stranded
  • DNA, Single-Stranded / metabolism*
  • Genes, Suppressor
  • Humans
  • Models, Molecular
  • Molecular Sequence Data
  • Phenylalanine / analogs & derivatives
  • Phenylalanine / metabolism
  • Phosphorylation
  • Phosphotyrosine / metabolism*
  • Protein Binding
  • Protein Conformation
  • Protein Processing, Post-Translational*
  • Proto-Oncogene Proteins c-abl / metabolism
  • Rad52 DNA Repair and Recombination Protein / chemistry
  • Rad52 DNA Repair and Recombination Protein / genetics
  • Rad52 DNA Repair and Recombination Protein / metabolism*
  • Recombinant Fusion Proteins / metabolism
  • Sequence Alignment
  • Sequence Homology, Amino Acid


  • Amino Acids
  • DNA, Single-Stranded
  • RAD52 protein, human
  • Rad52 DNA Repair and Recombination Protein
  • Recombinant Fusion Proteins
  • 4-carboxymethylphenylalanine
  • Phosphotyrosine
  • Phenylalanine
  • DNA
  • Proto-Oncogene Proteins c-abl