Delineating the position of rad4+/cut5+ within the DNA-structure checkpoint pathways in Schizosaccharomyces pombe

S. Harris
2003 Journal of Cell Science  
Introduction During cell growth and division the maintenance of genome integrity is aided by surveillance mechanisms that have been termed DNA structure checkpoint controls. These control mechanisms act to block cell-cycle progression in response to both DNA damage and the inhibition of DNA replication. The importance of DNA structure checkpoint mechanisms in normal growth control is best highlighted by the finding that mutations in certain mammalian checkpoint control genes (e.g. p53, ATM and
more » ... HK2) can lead to a predisposition to cancer and to other cellular pathologies (Hartwell and Kastan, 1994; Elledge, 1996; Carr, 2000) . The G2/M or DNA damage checkpoint arrests cell-cycle progression at the G2/M transition in the presence of damaged DNA, whereas the DNA replication checkpoint prevents entry into mitosis in the presence of stalled DNA replication forks and is referred to as the S-M checkpoint. Checkpoint-mediated responses to DNA damage are, to a large extent, conserved. Studies in the fission yeast Schizosaccharomyces pombe (S. pombe), in which these two major checkpoint pathways have been defined, have made a significant contribution to the understanding of the organization of the checkpoint pathways. Genetic and physiological experiments have revealed a core set of six proteins, collectively termed the 'checkpoint Rad' proteins (Rad1, Rad3, Rad9, Rad17, Rad26 and Hus1). 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. Rad17 associates with RFC subunits and, by analogy with the mechanism of eukaryotic DNA replication, may act to load the PCNA-like 9-1-1 complex onto chromatin (for a review, see O'Connell et al., 2000) . The regulated assembly of specific protein complexes onto the DNA in response to different checkpoint cues may prove a general and important feature of checkpoint regulation. Homologs of Rad3-Rad26 and Rad9-Rad1-Hus1 have been 3519 The fission yeast BRCT domain protein Rad4/Cut5 is required for genome integrity checkpoint responses and DNA replication. Here we address the position at which Rad4/Cut5 acts within the checkpoint response pathways. Rad4 is shown to act upstream of the effector kinases Chk1 and Cds1, as both Chk1 phosphorylation and Cds1 kinase activity require functional Rad4. Phosphorylation of Rad9, Rad26 and Hus1 in response to either DNA damage or inhibition of DNA replication are independent of Rad4/Cut5 checkpoint function. Further we show that a novel, epitope-tagged allele of rad4 + /cut5 + acts as a dominant suppressor of the checkpoint deficiencies of rad3 -, rad26and rad17mutants. Suppression results in the restoration of mitotic arrest and is dependent upon the remaining checkpoint Rad proteins and the two effector kinases. High-level expression of the rad4 + /cut5 + allele in rad17 mutant cells restores the nuclear localization of Rad9, but this does not fully account for the observed suppression. We conclude from these data that Rad4/Cut5 acts with Rad3, Rad26 and Rad17 to effect the checkpoint response, and a model for its function is discussed. Supplemental figure available online
doi:10.1242/jcs.00677 pmid:12865439 fatcat:j4plpptsifcnld35czjgbzpaui