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
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Research Support, Non-U.S. Gov't
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Research Support, U.S. Gov't, Non-P.H.S.
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Research Support, U.S. Gov't, P.H.S.
MeSH terms
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Adaptor Proteins, Signal Transducing
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Cell Cycle Proteins / genetics
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Cell Cycle Proteins / metabolism*
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Checkpoint Kinase 2
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DNA Damage
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Enzyme Activation
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Phosphoproteins / genetics
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Phosphoproteins / metabolism*
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Plasmids / genetics
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Protein Serine-Threonine Kinases / genetics
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Protein Serine-Threonine Kinases / metabolism*
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Recombinant Fusion Proteins / genetics
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Recombinant Fusion Proteins / metabolism*
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Saccharomyces cerevisiae / cytology
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Saccharomyces cerevisiae / genetics
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Saccharomyces cerevisiae / metabolism*
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Saccharomyces cerevisiae Proteins / genetics
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Saccharomyces cerevisiae Proteins / metabolism*
Substances
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Adaptor Proteins, Signal Transducing
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Cell Cycle Proteins
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LCD1 protein, S cerevisiae
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MRC1 protein, S cerevisiae
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Phosphoproteins
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Recombinant Fusion Proteins
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Saccharomyces cerevisiae Proteins
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rad9 protein
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Checkpoint Kinase 2
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Protein Serine-Threonine Kinases
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RAD53 protein, S cerevisiae