A Ddc2-Rad53 fusion protein can bypass the requirements for RAD9 and MRC1 in Rad53 activation

Mol Biol Cell. 2004 Dec;15(12):5443-55. doi: 10.1091/mbc.e04-07-0608. Epub 2004 Sep 29.

Abstract

Activation of Rad53p by DNA damage plays an essential role in DNA damage checkpoint pathways. Rad53p activation requires coupling of Rad53p to Mec1p through a "mediator" protein, Rad9p or Mrc1p. We sought to determine whether the mediator requirement could be circumvented by making fusion proteins between the Mec1 binding partner Ddc2p and Rad53p. Ddc2-Rad53p interacted with Mec1p and other Ddc2-Rad53p molecules under basal conditions and displayed an increased oligomerization upon DNA damage. Ddc2-Rad53p was activated in a Mec1p- and Tel1p-dependent manner upon DNA damage. Expression of Ddc2-Rad53p in Deltarad9 or Deltarad9Deltamrc1 cells increased viability on plates containing the alkylating agent methyl methane sulfonate. Ddc2-Rad53p was activated at least partially by DNA damage in Deltarad9Deltamrc1 cells. In addition, expression of Ddc2-Rad53p in Deltarad24Deltarad17Deltamec3 cells increased cell survival. These results reveal minimal requirements for function of a core checkpoint signaling system.

Publication types

  • Research Support, Non-U.S. Gov't
  • Research Support, U.S. Gov't, Non-P.H.S.
  • Research Support, U.S. Gov't, P.H.S.

MeSH terms

  • Adaptor Proteins, Signal Transducing
  • Cell Cycle Proteins / genetics
  • Cell Cycle Proteins / metabolism*
  • Checkpoint Kinase 2
  • DNA Damage
  • Enzyme Activation
  • Phosphoproteins / genetics
  • Phosphoproteins / metabolism*
  • Plasmids / genetics
  • Protein Serine-Threonine Kinases / genetics
  • Protein Serine-Threonine Kinases / metabolism*
  • Recombinant Fusion Proteins / genetics
  • Recombinant Fusion Proteins / metabolism*
  • Saccharomyces cerevisiae / cytology
  • Saccharomyces cerevisiae / genetics
  • Saccharomyces cerevisiae / metabolism*
  • Saccharomyces cerevisiae Proteins / genetics
  • Saccharomyces cerevisiae Proteins / metabolism*

Substances

  • Adaptor Proteins, Signal Transducing
  • Cell Cycle Proteins
  • LCD1 protein, S cerevisiae
  • MRC1 protein, S cerevisiae
  • Phosphoproteins
  • Recombinant Fusion Proteins
  • Saccharomyces cerevisiae Proteins
  • rad9 protein
  • Checkpoint Kinase 2
  • Protein Serine-Threonine Kinases
  • RAD53 protein, S cerevisiae