title: DNA replication checkpoint signaling depends on a Rad53-Dbf4 N-terminal interaction in Saccharomyces cerevisiae.
abstract: Dbf4-dependent kinase (DDK) and cyclin-dependent kinase (CDK) are essential to initiate DNA replication at individual origins. During replication stress, the S-phase checkpoint inhibits the DDK- and CDK-dependent activation of late replication origins. Rad53 kinase is a central effector of the replication checkpoint and both binds to and phosphorylates Dbf4 to prevent late-origin firing. The molecular basis for the Rad53-Dbf4 physical interaction is not clear but occurs through the Dbf4 N terminus. Here we found that both Rad53 FHA1 and FHA2 domains, which specifically recognize phospho-threonine (pT), interacted with Dbf4 through an N-terminal sequence and an adjacent BRCT domain. Purified Rad53 FHA1 domain (but not FHA2) bound to a pT Dbf4 peptide in vitro, suggesting a possible phospho-threonine-dependent interaction between FHA1 and Dbf4. The Dbf4-Rad53 interaction is governed by multiple contacts that are separable from the Cdc5- and Msa1-binding sites in the Dbf4 N terminus. Importantly, abrogation of the Rad53-Dbf4 physical interaction blocked Dbf4 phosphorylation and allowed late-origin firing during replication checkpoint activation. This indicated that Rad53 must stably bind to Dbf4 to regulate its activity.
authors.affiliation: Laboratory of Genome Integrity and Tumorigenesis, Van Andel Research Institute, Grand Rapids, MI 49503, USA.
authors.forename: Ying-Chou
authors.initials: YC
authors.lastname: Chen
authors.forename: Jessica
authors.initials: J
authors.lastname: Kenworthy
authors.forename: Carrie
authors.initials: C
authors.lastname: Gabrielse
authors.forename: Christine
authors.initials: C
authors.lastname: Hänni
authors.forename: Philip
authors.initials: P
authors.lastname: Zegerman
authors.forename: Michael
authors.initials: M
authors.lastname: Weinreich
mesh_terms: D020816:Amino Acid Motifs; D000595:Amino Acid Sequence; D018797:Cell Cycle Proteins / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D064447:Checkpoint Kinase 2 / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D004261:DNA Replication; D008969:Molecular Sequence Data; D010766:Phosphorylation; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D059807:S Phase Cell Cycle Checkpoints; D012441:Saccharomyces cerevisiae / Q000235:genetics* / Q000378:metabolism; D029701:Saccharomyces cerevisiae Proteins / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D015398:Signal Transduction
chemical_list: D018797:Cell Cycle Proteins; C088096:Dbf4 protein, S cerevisiae; D029701:Saccharomyces cerevisiae Proteins; D064447:Checkpoint Kinase 2; C067022:RAD53 protein, S cerevisiae
keywords: DDK; DNA replication; Dbf4; Rad53; replication checkpoint
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