An N-terminal domain of Dbf4p mediates interaction with both origin recognition complex (ORC) and Rad53p and can deregulate late origin firing.
The Dbf4Cdc7 kinase acts at the level of individual origins to promote the initiation of DNA replication. We demonstrate through both immunoprecipitation and two-hybrid assays that a domain comprising the first 296 aa of Dbf4p interacts with Orc2p and Orc3p subunits of the origin recognition complex (ORC). Given that the activation of Rad53 kinase in response to the DNA replication checkpoint leads to the release of Dbf4p from an ORC-containing chromatin fraction, we also examined interaction between Dbf4p and Rad53p. This same domain of Dbf4p binds specifically to the forkhead homology-associated (FHA) domains of Rad53p. Cell cycle arrest in G(2)M, provoked by the overexpression of the Dbf4 domain, is suppressed in a rad53 mutant. Moreover, its overexpression perturbs the regulation of late, but not early, origin firing in wild-type cells after treatment with hydroxyurea.
Duncker
Bernard P
BP
Department of Molecular Biology, University of Geneva, Quai Ernest-Ansermet 30, CH-1211 Geneva 4, Switzerland.
Shimada
Kenji
K
Tsai-Pflugfelder
Monika
M
Pasero
Philippe
P
Gasser
Susan M
SM
D001665:Binding Sites; D018797:Cell Cycle Proteins; D064447:Checkpoint Kinase 2; D004261:DNA Replication / Q000187:drug effects; D004271:DNA, Fungal / Q000096:biosynthesis; D004268:DNA-Binding Proteins / Q000737:chemistry / Q000378:metabolism*; D015966:Gene Expression Regulation, Fungal; D006918:Hydroxyurea / Q000494:pharmacology; D046911:Macromolecular Substances; D008957:Models, Genetic; D051738:Origin Recognition Complex; D011233:Precipitin Tests; D011485:Protein Binding; D025941:Protein Interaction Mapping; D017346:Protein Serine-Threonine Kinases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D017434:Protein Structure, Tertiary; D021122:Protein Subunits; D012441:Saccharomyces cerevisiae / Q000187:drug effects / Q000235:genetics / Q000378:metabolism; D029701:Saccharomyces cerevisiae Proteins / Q000737:chemistry* / Q000378:metabolism; D013329:Structure-Activity Relationship; D020798:Two-Hybrid System Techniques
D018797:Cell Cycle Proteins; D004271:DNA, Fungal; D004268:DNA-Binding Proteins; C088096:Dbf4 protein, S cerevisiae; D046911:Macromolecular Substances; D051738:Origin Recognition Complex; D021122:Protein Subunits; D029701:Saccharomyces cerevisiae Proteins; D064447:Checkpoint Kinase 2; D017346:Protein Serine-Threonine Kinases; C067022:RAD53 protein, S cerevisiae; D006918:Hydroxyurea