Visualization of the physical and functional interaction between hMYH and hRad9 by Dronpa bimolecular fluorescence complementation

BMC Mol Biol. 2014 Aug 15:15:17. doi: 10.1186/1471-2199-15-17.

Abstract

Background: Human MutY glycosylase homolog (hMYH), a component of the base excision repair pathway, is responsible for the generation of apurinic/apyrimidinic sites. Rad9-Rad1-Hus1 (9-1-1) is a heterotrimeric protein complex that plays a role in cell cycle checkpoint control and DNA repair. In humans, hMYH and 9-1-1 interact through Hus1 and to a lesser degree with Rad1 in the presence of DNA damage. In Saccharomyces pombe, each component of the 9-1-1 complex interacts directly with SpMYH. The glycosylase activity of hMYH is stimulated by Hus1 and the 9-1-1 complex and enhanced by DNA damage treatment. Cells respond to different stress conditions in different manners. Therefore, we investigated whether Rad9 interacted with hMYH under different stresses. Here, we identified and visualized the interaction between hRad9 and hMYH and investigated the functional consequences of this interaction.

Results: Co-IP and BiFC indicates that hMYH interacts with hRad9. As shown by GST-pull down assay, this interaction is direct. Furthermore, BiFC with deletion mutants of hMYH showed that hRad9 interacts with N-terminal region of hMYH. The interaction was enhanced by hydroxyurea (HU) treatment. mRNA and protein levels of hMYH and hRad9 were increased following HU treatment. A marked increase in p-Chk1 (S345) and p-Cdk2 (T14, Y15) was observed. But this phosphorylation decreased in siMYH- or siRad9-transfected cells, and more pronounced decrease observed in co-transfected cells.

Conclusions: Our data reveal that hRad9 interacts directly with N-terminal region of hMYH. This interaction is enhanced by HU treatment. Knockdown of one or both protein result in decreasing Chk1 and Cdk2 phosphorylation. Since both protein functions in the early detection of DNA damage, we suggest that this interaction occurs early in DNA damage pathway.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Cell Cycle Checkpoints
  • Cell Cycle Proteins / analysis
  • Cell Cycle Proteins / metabolism*
  • DNA Damage
  • DNA Glycosylases / analysis
  • DNA Glycosylases / metabolism*
  • Fluorescence Resonance Energy Transfer / methods
  • HEK293 Cells
  • Humans
  • Hydroxyurea / metabolism
  • Protein Interaction Domains and Motifs
  • Protein Interaction Mapping / methods
  • Protein Interaction Maps*

Substances

  • Cell Cycle Proteins
  • rad9 protein
  • DNA Glycosylases
  • mutY adenine glycosylase
  • Hydroxyurea