Abstract
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.
Highlights
DNA damage, from endogenous or environmental sources, can be disastrous for cell survival and proliferation if not accurately repaired
We have recently shown that the FHA domains are crucial for Rad53 DNA damage checkpoint functions as a rad53R70AR605A allele, containing disrupted phosphothreoninebinding sites in both FHA domains, displays severe hypersensitivity and greatly reduced Rad53 activation to DNA damage and replication blocks, similar to a rad53⌬ strain phenotype [30]
Genetic Analyses of the Interaction between the Rad53 FHA1 Domain and the Rad9 and Rad17 DNA Damage Sensor Pathways—We have previously shown that introduction of an R70A mutation into the endogenous RAD53 gene, which disrupts the FHA1 phosphothreonine-binding site without affecting domain folding, leads to moderately increased DNA damage sensitivity [32]
Summary
FHA domains contribute to the complex functions of Rad. We have recently shown that the FHA domains are crucial for Rad DNA damage checkpoint functions as a rad53R70AR605A allele, containing disrupted phosphothreoninebinding sites in both FHA domains, displays severe hypersensitivity and greatly reduced Rad activation to DNA damage and replication blocks, similar to a rad53⌬ strain phenotype [30]. In terms of requirements throughout the cell cycle, both FHA domains have specific roles during G2/M as inactivation of either FHA1 or FHA2 abolishes Rad activation and cell cycle arrest by the G2/M checkpoint, whereas the two FHA domains are largely redundant for Rad activation in asynchronous cells [30]. We demonstrate a novel function of the FHA1 domain by showing that it is required to link activated Rad to downstream effectors for its established function to prevent late origin firing as part of the replication checkpoint
Sign-in/Register to view highlights & summary Sign-in/Register to access full text options 
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a callDisclaimer: All third-party content on this website/platform is and will remain the property of their respective owners and is provided on "as is" basis without any warranties, express or implied. Use of third-party content does not indicate any affiliation, sponsorship with or endorsement by them. Any references to third-party content is to identify the corresponding services and shall be considered fair use under The CopyrightLaw.
