A Rad3–Rad26 complex responds to DNA damage independently of other checkpoint proteins

Abstract

The conserved PIK-related kinase Rad3 is required for all DNA-integrity-checkpoint responses in fission yeast. Here we report a stable association between Rad3 and Rad26 in soluble protein extracts. Rad26 shows Rad3-dependent phosphorylation after DNA damage. Unlike phosphorylation of Hus1, Crb2/Rhp9, Cds1 and Chk1, phosphorylation of Rad26 does not require other known checkpoint proteins. Rad26 phosphorylation is the first biochemical marker of Rad3 function, indicating that Rad3-related checkpoint kinases may have a direct role in DNA-damage recognition.

Delineating the position ofrad4+/cut5+ within the DNA-structure checkpoint pathways inSchizosaccharomyces pombe

“…A small amount of Rad26 phosphorylation is also detectable in the absence of DNA damage at 32°C. This is consistent with earlier data that demonstrated that Rad26 is phosphorylated in several Rad checkpoint-deficient backgrounds (Edwards et al, 1999). Checkpoint-dependent phosphorylation of Hus1 has also been reported after several hours' exposure to HU.…”

“…Activation and phosphorylation of Cds1 is S-phasespecific in response to either inhibition of DNA replication or DNA damage, and kinase activation correlates with phosphorylation of the protein , providing a useful marker for the S-M checkpoint. Rad9 and Rad26 are also phosphorylated in response to radiationinduced DNA damage and Hus1 is phosphorylated in response to both DNA damage and prolonged inhibition of DNA replication (Kostrub et al, 1998;Edwards et al, 1999;Caspari et al, 2000a;Caspari et al, 2000b). Ultimately, all of these phosphorylation events have been demonstrated to be Rad3-dependent and this has enabled hierarchical relationships between fission yeast checkpoint proteins to be determined.…”

“…These proteins are fundamental to both pathways and appear necessary for the receipt and transmission of the checkpoint signal (Al-Khodairy and Carr, 1992;Enoch et al, 1992;O'Connell et al, 2000). As yet, the precise mechanism(s) by which these proteins act to achieve this goal is unclear, although recent studies have indicated the presence of discrete intracellular complexes between Rad3 and Rad26, between Rad17 and the four small subunits of replication factor C (RFC) and between Rad9, Rad1 and Hus1 (Edwards et al, 1999;Shimada et al, 1999;Kostrub et al, 1998;Caspari et al, 2000a). The Rad3-Rad26 complex functions as a PI3-related protein kinase and the Rad1-dependent complex (known as the 9-1-1 complex) is related to the proliferating cell nuclear antigen (PCNA) sliding clamp.…”

“…In the event of DNA damage occurring in S phase, the Rad17 and 9-1-1 complexes appear to be required for Rad3 phosphorylation of Rad26, suggesting that the loading or activation of Rad3 requires a function of the RFC-and PCNArelated proteins. Thus under these circumstances they may formally act above the Rad3 kinase (Edwards et al, 1999;O'Connell et al, 2000).…”

An ATM- and ATR-dependent checkpoint inactivates spindle assembly by targeting CEP63

“…ATR signalling is also induced by bulky DNA adducts, lesions resulting from ultraviolet radiation (UV), such as pyrimidine dimers and the 6-(1,2)-dihydro-2-oxo-4-pyrimidinyl-5-methyl-2,4-(1H,3H)-pyrimidinediones (Unsal-kacmaz et al, 2002). In humans, ATR is stably associated with ATRIP (ATR/ATRIP), this is also the case in yeast (Mec1/Dcd2 in S.cerevisiae (budding yeast) and Rad3/Rad56 in Schizosaccharomyces Pombe (S.pombe, fission yeast) (Cortez et al, 2001;Unsal-kacmaz and Sancar, 2004;Paciotti et al, 2000;Edwards et al, 1999). ATR is thought to be constitutively active during unstressed DNA replication, thus it is believed that during S phase substrate reactions are reliant on cellular localisation (Niida and Nakanishi, 2006).…”