Rad53 kinase activation-independent replication checkpoint function of the N-terminal forkhead-associated (FHA1) domain.
Saccharomyces cerevisiae Rad53 has crucial functions in many aspects of the cellular response to DNA damage and replication blocks. To coordinate these diverse roles, Rad53 has two forkhead-associated (FHA) phosphothreonine-binding domains in addition to a kinase domain. Here, we show that the conserved N-terminal FHA1 domain is essential for the function of Rad53 to prevent the firing of late replication origins in response to replication blocks. However, the FHA1 domain is not required for Rad53 activation during S phase, and as a consequence of defective downstream signaling, Rad53 containing an inactive FHA1 domain is hyperphosphorylated in response to replication blocks. The FHA1 mutation dramatically hypersensitizes strains with defects in the cell cycle-wide checkpoint pathways (rad9Delta and rad17Delta) to DNA damage, but it is largely epistatic with defects in the replication checkpoint (mrc1Delta). Altogether, our data indicate that the FHA1 domain links activated Rad53 to downstream effectors in the replication checkpoint. The results reveal an important mechanistic difference to the homologous Schizosaccharomyces pombe FHA domain that is required for Mrc1-dependent activation of the corresponding Cds1 kinase. Surprisingly, despite the severely impaired replication checkpoint and also G(2)/M checkpoint functions, the FHA1 mutation by itself leads to only moderate viability defects in response to DNA damage, highlighting the importance of functionally redundant pathways.
St. Vincent's Institute of Medical Research, Department of Medicine, The University of Melbourne, Fitzroy, Victoria, Australia. bpike@svi.edu.au
Brietta L
BL
Pike
Nora
N
Tenis
Jörg
J
Heierhorst
D001665:Binding Sites; D018797:Cell Cycle Proteins / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D064447:Checkpoint Kinase 2; D004261:DNA Replication / Q000502:physiology*; D004268:DNA-Binding Proteins; D051858:Forkhead Transcription Factors; D018816:Genes, cdc / Q000502:physiology; D009154:Mutation; D009687:Nuclear Proteins / Q000737:chemistry / Q000235:genetics; D010749:Phosphoprotein Phosphatases / Q000378:metabolism; D010766:Phosphorylation; D010769:Phosphothreonine / Q000378:metabolism; D000071636:Protein Phosphatase 2C; D017346:Protein Serine-Threonine Kinases / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D017434:Protein Structure, Tertiary; D018741:Replication Origin; D012441:Saccharomyces cerevisiae / Q000201:enzymology* / Q000235:genetics; D029701:Saccharomyces cerevisiae Proteins / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D014157:Transcription Factors / Q000737:chemistry / Q000235:genetics
D018797:Cell Cycle Proteins; D004268:DNA-Binding Proteins; D051858:Forkhead Transcription Factors; D009687:Nuclear Proteins; C501395:RAD17 protein, S cerevisiae; D029701:Saccharomyces cerevisiae Proteins; D014157:Transcription Factors; D010769:Phosphothreonine; C069930:rad9 protein; D064447:Checkpoint Kinase 2; D017346:Protein Serine-Threonine Kinases; C067022:RAD53 protein, S cerevisiae; D010749:Phosphoprotein Phosphatases; D000071636:Protein Phosphatase 2C