Interaction between Rad9-Hus1-Rad1 and TopBP1 activates ATR-ATRIP and promotes TopBP1 recruitment to sites of UV-damage

DNA Repair (Amst). 2014 Sep;21:1-11. doi: 10.1016/j.dnarep.2014.05.001. Epub 2014 May 27.

Abstract

The checkpoint clamp Rad9-Hus1-Rad1 (9-1-1) interacts with TopBP1 via two casein kinase 2 (CK2)-phosphorylation sites, Ser-341 and Ser-387 in Rad9. While this interaction is known to be important for the activation of ATR-Chk1 pathway, how the interaction contributes to their accumulation at sites of DNA damage remains controversial. Here, we have studied the contribution of the 9-1-1/TopBP1 interaction to the assembly and activation of checkpoint proteins at damaged DNA. UV-irradiation enhanced association of Rad9 with chromatin and its localization to sites of DNA damage without a direct interaction with TopBP1. TopBP1, as well as RPA and Rad17 facilitated Rad9 recruitment to DNA damage sites. Similar to Rad9, TopBP1 also localized to sites of UV-induced DNA damage. The DNA damage-induced TopBP1 redistribution was delayed in cells expressing a TopBP1 binding-deficient Rad9 mutant. Pharmacological inhibition of ATR recapitulated the delayed accumulation of TopBP1 in the cells, suggesting that ATR activation will induce more efficient accumulation of TopBP1. Taken together, TopBP1 and Rad9 can be independently recruited to damaged DNA. Once recruited, a direct interaction of 9-1-1/TopBP1 occurs and induces ATR activation leading to further TopBP1 accumulation and amplification of the checkpoint signal. Thus, we propose a new positive feedback mechanism that is necessary for successful formation of the damage-sensing complex and DNA damage checkpoint signaling in human cells.

Keywords: Checkpoint control; DNA damage response; DNA repair; DNA replication; Protein–protein interactions.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Adaptor Proteins, Signal Transducing / genetics
  • Adaptor Proteins, Signal Transducing / metabolism*
  • Ataxia Telangiectasia Mutated Proteins / genetics
  • Ataxia Telangiectasia Mutated Proteins / metabolism
  • Carrier Proteins / genetics
  • Carrier Proteins / metabolism*
  • Cell Cycle Proteins / genetics
  • Cell Cycle Proteins / metabolism*
  • Checkpoint Kinase 1
  • Chromatin / genetics
  • Chromatin / metabolism
  • DNA Damage*
  • DNA-Binding Proteins / genetics
  • DNA-Binding Proteins / metabolism*
  • Exonucleases / genetics
  • Exonucleases / metabolism*
  • HeLa Cells
  • Humans
  • Nuclear Proteins / genetics
  • Nuclear Proteins / metabolism*
  • Protein Binding
  • Protein Kinases / genetics
  • Protein Kinases / metabolism
  • Protein Transport
  • Ultraviolet Rays

Substances

  • ATRIP protein, human
  • Adaptor Proteins, Signal Transducing
  • Carrier Proteins
  • Cell Cycle Proteins
  • Chromatin
  • DNA-Binding Proteins
  • HUS1 protein, human
  • Nuclear Proteins
  • Rad17 protein, human
  • TOPBP1 protein, human
  • rad9 protein
  • Protein Kinases
  • ATR protein, human
  • Ataxia Telangiectasia Mutated Proteins
  • CHEK1 protein, human
  • Checkpoint Kinase 1
  • Exonucleases
  • Rad1 protein, human