53BP1 exchanges slowly at the sites of DNA damage and appears to require RNA for its association with chromatin

Fiona Pryde, Shirin Khalili, Kathryn Robertson, Jim Selfridge, Ann-Marie Ritchie, David W Melton, Denis Jullien, Yasuhisa Adachi

Research output: Contribution to journalArticlepeer-review

Abstract

53BP1 protein is re-localized to the sites of DNA damage after ionizing radiation (IR) and is involved in DNA-damage-checkpoint signal transduction. We examined the dynamics of GFP-53BP1 in living cells. The protein starts to accumulate at the sites of DNA damage 2-3 minutes after damage induction. Fluorescence recovery after photobleaching experiments showed that GFP-53BP1 is highly mobile in non-irradiated cells. Upon binding to the IR-induced nuclear foci, the mobility of 53BP1 reduces greatly. The minimum (M) domain of 53BP1 essential for targeting to IR induced foci consists of residues 1220-1703. GFP-M protein forms foci in mouse embryonic fibroblast cells lacking functional endogenous 53BP1. The M domain contains a tandem repeat of Tudor motifs and an arginine- and glycine-rich domain (RG stretch), which are often found in proteins involved in RNA metabolism, the former being essential for targeting. RNase A treatment dissociates 53BP1 from IR-induced foci. In HeLa cells, dissociation of the M domain without the RG stretch by RNase A treatment can be restored by re-addition of nuclear RNA in the early stages of post-irradiation. 53BP1 immunoprecipitates contain some RNA molecules. Our results suggest a possible involvement of RNA in the binding of 53BP1 to chromatin damaged by IR.

Original languageEnglish
Pages (from-to)2043-55
Number of pages13
JournalJournal of Cell Science
Volume118
Issue numberPt 9
DOIs
Publication statusPublished - 1 May 2005

Keywords

  • Amino Acid Sequence
  • Animals
  • Chromatin
  • DNA Damage
  • DNA Repair
  • Fibroblasts
  • Fluorescence Recovery After Photobleaching
  • Green Fluorescent Proteins
  • HeLa Cells
  • Homozygote
  • Humans
  • Immunoprecipitation
  • Intracellular Signaling Peptides and Proteins
  • Mice
  • Mice, Transgenic
  • Microscopy, Fluorescence
  • Molecular Sequence Data
  • Mutation
  • Phosphoproteins
  • Plasmids
  • Protein Binding
  • Protein Structure, Tertiary
  • RNA
  • Ribonuclease, Pancreatic
  • Ribonucleases
  • Signal Transduction
  • Time Factors

Fingerprint Dive into the research topics of '53BP1 exchanges slowly at the sites of DNA damage and appears to require RNA for its association with chromatin'. Together they form a unique fingerprint.

Cite this